Shroom3 is essential for cell shape change during gut morphogenesis. (A) Diagram of a transverse section of the stage 32 Xenopus embryo. (B) Shroom3 expression in the endoderm at stage 32. Note the robust Shroom3 expression (arrow) in the archenteron floor and roof. (C) α-tubulin staining of the archenteron floor in a control embryo. The archenteron floor displays a V-shaped morphology. (D) Archenteron floor morphology is disrupted and microtubule (MT) arrays are reduced in cells lacking Shroom3. We observed a less acute archenteron floor angle in Shroom3 MO-injected embryos (P=0.001, Mann-Whitney U-test). Moreover, archenteron floor thickness is reduced from 68±1 μm (mean ± s.e.m.; n=13) in control embryos to 59±1 μm (n=21) (P=0.0003) in Shroom3 MO-injected embryos. (E) γ-tubulin staining of the archenteron floor in a control embryo. (F) γ-tubulin accumulation is reduced in archenteron floor cells lacking Shroom3 function. (G) ZO-1 staining of tight junctions in archenteron floor cells. (H) The apical surface of archenteron floor cells is less constricted in the Shroom3 morphant embryo. ZO-1 localization is not affected. Scale bars: 10 μm.

Pitx1 and Shroom3 are co-expressed in the developing gut. (A-C) Expression patterns of (A) Pitx1 and (B) Shroom3 in a transverse section of the stage 32 Xenopus embryo, as schematized in C. Both Shroom3 and Pitx1 are expressed in the archenteron roof and floor (arrowheads). (D,E) At stage 44, a ventral view of dissected guts reveals that both (D) Pitx1 and (E) Shroom3 are expressed in the foregut (arrows).

Pitx1 controls Shroom3 expression in the developing gut. (A,B) Transverse sections showing Shroom3 expression in (A) a control Xenopus embryo and (B) the Pitx1 MO-injected embryo, where Shroom3 expression is reduced in the archenteron floor (arrows). The regions indicated by the arrows in A and B approximate to those shown at higher magnification in C,E and D,F, respectively. (C) α-tubulin staining of control archenteron floor cells. (D) α-tubulin staining of archenteron floor cells lacking Pitx1. The archenteron floor fails to take on its deep V-shaped morphology, showing an average archenteron floor angle 132±4° (mean ± s.e.m.; n=22), compared with 62±5° (n=16) (P<0.0001) in control embryos. Moreover, the archenteron thickness is reduced from 90±3 μm (mean ± s.e.m.; n=13) to 68±3 μm (n=17) (P<0.001). (E) γ-tubulin staining of the archenteron floor in the control embryo. (F) γ-tubulin accumulates less in archenteron floor cells lacking Pitx1. (G) ZO-1 staining of control archenteron floor cells. (H) The apical surface of archenteron floor cells is less constricted in the Pitx1 knockdown embryo, whereas ZO-1 localization is not affected. Scale bars: 10 μm.

Pitx1 is essential for Xenopus gut morphogenesis. (A,A′,C,C′) Ventral (A,A′) and lateral (C,C′) views of a stage 44 dissected control gut reveal Shroom3 expression in the foregut (arrow in A). In the diagrams, Shroom3 expression is represented in purple and normal gut looping by the black line. (B,B′,D,D′) Ventral (B,B′) and lateral (D,D′) views of dissected guts lacking Pitx1 reveal reduced Shroom3 expression in the foregut (arrow in B). The Pitx1 morphant displays a failure of looping (red line) in the foregut, corresponding to regions of disrupted Shroom3 expression.

Ectopic Pitx1 activates Shroom3 expression. (A) Stage 19 control Xenopus embryo. (B) Ectopic expression of Pitx1 in the epidermis induces ectopic pigment accumulation, similar to that induced by Shroom3 expression (Haigo et al., 2003; Lee et al., 2007). (C) Shroom3 expression pattern in a control embryo. (D) Ectopic Pitx1 expression activates ectopic Shroom3 expression in the epidermis. (E,F) Control (E) and Pitx3-injected (F) embryos. Ectopic Pitx3 also induces pigment accumulation. (G) Control Shroom3 expression pattern. (H) Ectopic Pitx3 expression activates ectopic Shroom3 expression in the epidermis. (I) Experimental scheme for the animal cap assay. Pitx1 mRNA (100 pg) was injected into the animal pole at the 4-cell stage. Animal caps were dissected at stage 8 and cultured to stage 16. (J) Shroom3 and Shroom1 expression levels in whole embryos, control caps, and Pitx1-injected caps were analyzed by RT-PCR using Xag1 as a positive control and Ef1α as a loading control. Shroom3 expression is induced following Pitx1 expression. (K) Shroom3 expression is induced following Pitx3 expression in animal caps.

Pitx1 directly activates Shroom3 transcription. (A) Experimental scheme. Pitx1-GR mRNA was injected into the animal pole at the 4-cell stage. Animal caps were dissected at stage 8 and cultured. At stage 13, caps were first treated with cycloheximide (CHX) for 10 minutes and then treated with dexamethasone (DEX) and cultured to stage 16. (B) Shroom3 and Xag1 expression are induced by active Pitx1. Shroom3 is consistently induced in the presence of both CHX and DEX, although Xag1 is not activated by DEX in the presence of CHX. (C,D) The same experiment described in A, but using (C) Pitx2-GR and (D) Pitx3-GR mRNA. Similar data were obtained as for Pitx1-GR.

A Pitx1-responsive enhancer is located 12 kb upstream of the Shroom3 transcription start site. (A) Three Pitx1 consensus binding sites are located in a cluster ∼12 kb upstream of the Xenopus Shroom3 transcription start site. (B) The fragment containing these three sites (shr3-12k) was cloned into the pGL3-Cska minimal promoter-luciferase vector. Embryos at the 4-cell stage were injected in each blastomere with luciferase reporter constructs and Pitx1 mRNA, as indicated in C. Embryos were collected at stage 12 for luciferase assays. (C) Normalized luciferase activity. Together with Pitx1 expression, inclusion of the shr3-12k region increases luciferase activity ∼18-fold above the basal level observed with the minimal promoter alone.