Whole mount expression pattern of <i>Six2</i>-cre in E12.5 kidney and stomach.

<p>(A) <i>Six2</i>-cre (GFP, green) is expressed in the cap metanephric mesenchyme (mm) surrounding the ureteric bud tips. Wnt9b expression and canonical Wnt signaling is highest in the ureteric bud which is labeled with cytokeratin (red). (B) In the stomach, <i>Six2</i>-cre is expressed in the hindstomach and pyloric region. Canonical Wnt signaling activity is downregulated in this region <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043098#pone.0043098-Kim1" target="_blank">[13]</a>.</p

Lef1 expression exhibits dose-dependent effects in the <i>Wnt9b</i> and stabilized β-catenin alleles.

<p>(A–C) Lef1 expression (green) is low in the self-renewing cap metanephric mesenchyme cells (mm) and upregulated in the differentiating renal vesicle structures (rv). NCAM expression is also increased as the cap mm differentiates, but is not expressed in the ureteric bud (ub). (D–I) <i>Wnt9b</i> transgenic kidneys have increased Lef1 staining which is further upregulated in the ectopic vesicles seen in the stabilized β-catenin allele. Notably, not all ectopic vesicles in the stabilized β-catenin allele are NCAM positive, suggesting abnormal specification of these differentiated structures.</p

Duodenogastric reflux and pyloric sphincter abnormalities in <i>Six2</i>-cre^tg/+, <i>Wnt9b</i>^tg/+ double heterozygotes.

<p>(A, B) Yellow amniotic fluid refluxed into the stomach of E18.5 double heterozygotes, but was properly retained in the intestine in control animals. A lack of constriction (arrow) and an increased diameter (inset) suggests that the reflux is due to a malformed pyloric sphincter. (C, D) Whole mount staining for <i>Nephrocan</i> mRNA delineates a thin band demarking the sphincter at E16.5 that is expanded in the double heterozygotes. (E, F) Smooth muscle actin staining in sections from control adult stomach demonstrates a thick muscular layer that leads to the sphincter. Double heterozygotes exhibit a thickened muscular layer; however it does not coalesce at the point of highest constriction. Vertical lines mark the transition from antral stomach (left side) to duodenum (right side) in E–H. (G, H) Alcian blue stains mucosal cells in the antral stomach in control sections and these cells are absent in the double heterozygotes. Mucosal cells in the intestine are stained normally. (I, J) Staining for the H⁺/K⁺ ATPase detects parietal cells in the forestomach that are excluded from the distal stomach. These cells are found throughout the distal stomach and pylorus in double heterozygotes. Sections depicted in panels I and J highlight the transition between forestomach and distal stomach (diagonal line) and do not include the duodenum.</p

Kidney cysts in double transgenics can be found throughout all segments of the nephron.

<p>(A–D) H&E staining of adult kidneys demonstrates numerous cystic dilatations in the cortex and outer medulla in <i>Six2-cre^tg/+</i>, <i>Wnt9b^tg/+</i> double transgenics and a normal kidney architecture in <i>Wnt9b^tg/+</i> controls. (E–H) Cysts were derived from proximal tubules (LTL-positive) and distal loop of Henle (THP-positive). DBA-positive and Aquaporin 2- positive collecting duct cysts were also identified in smaller number. Since <i>Six2</i>-cre is not expressed in the collecting duct, these cysts are produced through signaling from neighboring cells or secondary to dilatation of more proximal segments.</p

Pyloric gene expression exhibits dose-dependent effects in the <i>Wnt9b</i> and stabilized β-catenin alleles.

<p>Whole mount in situ analysis of <i>Grem1</i>, <i>Nkx2.5</i>, and <i>Gata3</i> expression in E12.5 stomachs. All three genes are restricted to the pyloric sphincter in wild-type stomachs (<i>Six2</i>-cre^tg/+). <i>Grem1</i> and <i>Gata3</i> show decreased expression in <i>Wnt9b</i> transgenics (<i>Six2-cre^tg/+</i>, <i>Wnt9b^tg/+</i>) that is further downregulated in the stabilized β-catenin allele (<i>Six2-cre^tg/+</i>, <i>Ctnnb1^ex3/+</i>). <i>Nkx2.5</i> expression is expanded into the stomach in <i>Wnt9b</i> transgenics and further upregulated in the stabilized β-catenin allele. <i>Nkx2.5</i> expression that can be seen at the upper right of the stomach is found in the spleen on the opposite side.</p

Transgene and GFP expression depends on cre in cells and tissues.

<p>(A) Schematic of the transgene construct showing the <i>β-actin</i> promoter (β-actin^pro), the stop cassette (STOP) flanked by lox P sites (triangles), the epitope-tagged <i>Wnt9b</i> cDNA (Wnt9bFLU), an internal ribosomal entry site (IRES) and green fluorescent protein cDNA (GFP). (B) Epitope-tagged Wnt9b protein is expressed when the transgene is introduced into Cos-1 cells (lanes 1, 2) and E11.5 transgenic embryos (lanes 3–6) only when cre recombinase is present (cre +). There is a higher molecular weight background band present in the tissue lysates that is not altered by the presence of cre. Lane 7 is empty. A positive control vector that lacks the lox-STOP cassette expresses Wnt9b-FLU in the absence of cre (lane 8). Brightfield (C–D) and fluorescence (E, F) images of <i>Wnt9b</i> transgenic E11.5 embryos with (D,F) and without (C,E) <i>β-actin</i>-cre demonstrate GFP expression and delayed embryogenesis for the <i>Wnt9b</i>^tg/+, <i>β-actin</i>-cre^tg/+ embryos.</p