Six2-null embryos exhibit duodenogastric reflux and mucosal overgrowth. (A) At E18.5, the wild-type stomach contains a functional pyloric sphincter (PS; arrow) to prevent the reflux of amniotic fluid from the small intestine (SI) to the stomach. (B) The Six2-null stomach lacks a functional PS (arrow), thereby allowing reflux of the fluid (yellow content). (C, E) At this same stage, H & E staining of the wild-type stomach shows the region of the forming PS (arrows), which includes a thickened circular smooth muscle layer that constricts the gut tube at the junction of the stomach and SI. (D, F) The Six2-mutant gut tube lacks this thickened smooth muscle layer and the constriction that normally occurs at the corresponding level where the PS (arrows) should have formed. (G) At E18.5, the wild-type glandular stomach consists of primitive mucosal glands. (H) The Six2-null stomach exhibits overgrowth of the glands at this stage (compare arrows in G and H). Scale bars, 100 µm.

Six2 is required to maintain a Bmp4-free territory in the prospective pyloric sphincter (PS) region. (A) Normally at E11.5, Bmp4 is expressed in the mesenchyme surrounding the epithelium of the presumptive duodenum (arrowhead) and is absent from the mesoderm of the presumptive PS region (arrow). (B) This expression pattern is not obviously affected in the Six2-null gut tube. (C, C’) At E12.5, Bmp4 expression is excluded from the PS territory (arrow) in wild-type embryos. (D, D’) In Six2^−/− littermates, expression of Bmp4 has ectopically expanded into the prospective PS region (arrow). (E) Expression of Bmpr1b is detected in the wild-type mesenchyme of the developing glandular stomach and PS region (arrow). (F) This expression pattern appears unaffected in the Six2-null stomach. Bmpr1b expression remains in the PS territory (arrows) of E12.5 wild-type (G) and Six2-null (H) stomachs. Scale bars, 100 µm.

Model of Six2 function during mouse pyloric sphincter development. Based on the results presented here and those published for chick PS development, we propose a model for PS formation in the mouse. Six2 activity in the GS region of the developing stomach functions to suppress expression of Bmp4 in the PS territory. It is possible that this regulation of the Bmp4 pathway is accomplished through transcriptional regulation of another unidentified factor. Other PS markers (Gremlin, Nkx2.5, and Sox9) are regulated downstream of Six2 activity either by an unidentified factor or by the Bmp4 pathway. Gremlin can further abrogate Bmp signaling to control smooth muscle thickness.

Six2 is expressed in the mesoderm of the posterior stomach. (A) At E9.5, Six2 is expressed in the splanchnic mesoderm of the mouse stomach anlage (arrow). (B) By E10.5, Six2 is expressed in the mesoderm of the posterior stomach (arrow). (C) Expression is seen in the presumptive glandular stomach primordium at E11.5. (D, E) At E12.5, Six2 becomes restricted to the mesenchyme of the antral region of the posterior stomach (arrows); no expression is observed in the epithelial layer (arrowhead). (F) At E14.5, Six2 expression remains in the antrum, just anterior to the pyloric sphincter (arrowhead). Small intestine, SI; Scale bars, 100 µm.

The lack of Six2 activity disrupts the initial steps of pyloric sphincter (PS) formation. (A, C) Expression of α-smooth muscle actin (α-SMA) in the E14.5 wild-type stomach reveals the thickening of the smooth muscle layer and the constriction of the gut tube at the level of the developing PS (arrows). (B, D) In the E14.5 Six2-null stomach, the α-SMA⁺ layer fails to thicken, and the constriction of the gut tube at the boundary of the stomach and small intestine (arrows) does not form. (E) At E18.5, the wild-type stomach shows the characteristic thickening of the smooth muscle layer and constriction of the gut tube in the PS territory (arrow). (F) The Six2-null stomach shows no evidence of PS formation at the stomach-SI junction (arrow). (G, H) The thickness of the smooth muscle layer (arrows) in the remainder of the Six2-null stomach (H) is similar to that of the wild-type stomach (G), as demonstrated by representative sections of the glandular stomach. Scale bars, 100 µm.

Expression of pyloric sphincter (PS) markers is aberrant in the Six2-null stomach. (A) Nkx2.5 is expressed in the E12.5 wild-type PS territory (arrow). (B) However, in the prospective PS territory of E12.5 Six2^−/− littermates, the domain of Nkx2.5 expression (arrow) is smaller. (C) At E14.5, Nkx2.5 expression remains in the wild-type presumptive PS territory (arrow). (D) Nkx2.5 expression appears normal in the Six2-null stomach at this stage. Sox9 is expressed in the mesenchyme of the prospective PS region (arrows) and in the epithelium of the stomach (arrowheads) at E12.5 (E) and E14.5 (G). In the absence of Six2, the domain of mesodermal cells (arrows) expressing Sox9 is smaller at E12.5 (F) and absent at E14.5 (H); its pattern of expression is normal in the stomach epithelium at both stages (arrowheads). Gremlin is also expressed in the mesenchyme of the presumptive wild-type PS region (arrows) at E12.5 and E14.5 (I, K). The territory of mesenchymal cells expressing Gremlin (arrows) is smaller in the Six2^−/− stomach at E12.5 (J) and E14.5 (L). Scale bars, 100 µm.