Sinha T , Lin L , Li D , Davis J , Evans S , Wynshaw-Boris A , Wang J .

DP1 HL117649 NHLBI NIH HHS , HL109130 NHLBI NIH HHS , HL117649 NHLBI NIH HHS , HL119967 NHLBI NIH HHS , R01 HL109130 NHLBI NIH HHS , R01 HL119967 NHLBI NIH HHS

             Wnt signaling pathway
             establishment of cell polarity

	Fig. 1. Generation and characterization of Wnt11-CreER BAC transgene. (A) Schematic diagram of BAC clone RP23-122D14 that contains the 19.6 kb mouse Wnt11 locus, and 102.7 kb and 74.5 kb genomic sequence flanking the 5′ and 3′ ends of Wnt11, respectively. (B, C) A CreER-T2 targeting cassette was used to partially replace the first coding exon (exon3) of Wnt11 by BAC recombineering to create the tamoxifen-inducible Wnt11-CreER BAC transgene. (D–I) Wnt11-CreER BAC transgenic mice were crossed with Cre reporter R26R-LacZ and the expression pattern of Wnt11-CreER was determined by X-gal staining to examine Cre-induced LacZ expression 6–12 h after tamoxifen injection. (D) When tamoxifen was injected at E6.25 and X-gal staining was performed 6 h later at E6.5, labeled cells were observed in the proximal extra-embryonic region near the ectoplacental cone, and in the posterior region of the embryo adjacent to the embryonic/extraembryonic border and the primitive streak. (E–G) Tamoxifen injection at E7.25 and X-gal staining at E7.5 resulted in labeled cells in the node (arrows in (E–G)) and the posterior end of the embryo, surrounding the primitive streak and at the base of the allantois. (H, I) Tamoxifen administration at E9.0 and X-gal staining at E9.5 resulted in labeled cells in the posterior trunk/tailbud region and the nephric duct (ND, arrow in I) and in the outflow tract and the second pharyngeal arch anteriorly (H). A (anterior); EPC (ectoplacental cone); HT (heart tube); ND (nephric duct (ND); P (posterior); PM (pharyngeal mesoderm).
	Fig. 2. Cells expressing Wnt11-CreER at the onset of gastrulation contribute to the endodermal and endothelial lineages. Wnt11-CreER mediated recombination and activation of tdTomato expression were induced by a single dose of tamoxifen administration at E6.0, and embryos were harvested and analyzed at E8.5. (A) Epi-fluorescence of tdTomato alone, and (B) merged with brightfield. Saggital (C–F′) and transverse (G–J″) sections of the embryos co-stained with DAPI (nucleus) revealed that tdTomato expressing cells (red) were present along the endoderm from anterior (F and magnified view in F′, red arrows) to the posterior (F, J and magnified view in J′). A small number of tdTomato expressing cells also co-expressed the endothelial marker PECAM (D, F H, J and J″) and were present in the dorsal aorta in the embryo proper (green arrow in F; magnified in F′) and as a few clusters in the yolk sac vasculature (green arrow in J; magnified in J″). The dotted line in (B) indicates the plane of sectioning in G–J″. ant NT (anterior neural tube); DA (dorsal aorta); En (endoderm endoderm); Post. en (posterior endoderm); post NT (posterior neural tube); YSV (yolk sac vasculature).
	Fig. 3. Cells expressing Wnt11-CreER at the mid streak stage contribute to the endodermal and mesodermal lineages. Wnt11-CreER mediated recombination and activation of tdTomato expression were induced by a single dose of tamoxifen administration at E6.5, and embryos were harvested and analyzed at E8.5. (A, B) Epi-fluorescence of tdTomato alone, and (A′, B′) merged with brightfield. (C–F) Embryo sections co-stained with PECAM (green) and DAPI (blue) revealed that tdTomato (red) expressing cells occupied many more patches of the vasculature in the yolk sac (C) and the allantois (F) when compared to E6.0 tamoxifen induced embryos (Fig. 2F). Increased contribution of tdTomato expressing cells to the endothelial lineage was also evident in the dorsal aorta (green arrows in (D) and (D′)). TdTomato expressing cells also occupied the entire endoderm, from the anterior to the posterior endoderm (red arrows in (C) and (E)). Dotted lines in (A′) and (B′) indicate the plane of sectioning in (C–E). ant NT (anterior neural tube); DA (dorsal aorta); FG (foregut); post NT (posterior neural tube); YSV (yolk sac vasculature).
	Fig. 4. Contribution of E6.5 Wnt11-CreER expressing cells to the endothelium and endodermally derived internal organs in E12.5 embryos. Wnt11-CreER mediated recombination and activation of tdTomato expression were induced by a single dose of tamoxifen administration at E6.5, and embryos were harvested and analyzed at E12.5. (A–D″) Whole mount epi-fluorescent analysis showed that tdTomato expressing cells line the yolk-sac vasculature (A and A′), and co-expressed the endothelial marker PECAM (B, B′ and B″). Bisection of the placenta revealed widespread tdTomato expression in the placental labyrinth (C and C′). In the placenta, tdTomato expression was present mainly in the endothelial cells in the fetal vasculature (D′ and D″) as well as the supporting chorio-allanatoic mesenchyme (yellow arrows in D and D″) surrounding the larger placental vessels. (E–H″) In the embryo proper, tdTomato expressing cells contributed to the endodermally derived internal organs, namely the lungs and the gastro-intestinal tract. (E, E′, G, G′) Whole mount epifluorescent analysis. Sectioning along the indicated planes in E′ and G′ (dashed white lines) revealed that the tdTomato expressing cells were present mostly in the epithelial lining of the lungs (white arrows in F and F′; magnified view in F″) and stomach (white arrow in H and H′; magnified view in H″). Eso: esophagus, GIT: gastro-intestinal tract.
	Fig. 5. Cells expressing Wnt11-CreER at late streak stage contribute to the posterior endoderm and to distinct mesodermal tissues. Wnt11-CreER mediated recombination and activation of tdTomato expression were induced by a single dose of tamoxifen administration at E7.5, and embryos were harvested and analyzed at E8.75. (A) Epi-fluorescence of tdTomato alone, and (A′) merged with brightfield. (B) Saggital section of the embryonic region (yellow box in (A′) revealed the contribution of tdTomato expressing cell to the endoderm (red arrow in B), the inter-somitic vessels (ISV, green arrows in B), the dorsal aorta (DA) and the posterior lateral plate mesoderm (tdTomato-positive cells between dorsal aorta and endoderm). (C–C″) Transverse sectioning of these embryos along the dotted line in (A′) and co-staining with nuclear marker DAPI (blue) and endothelial cell marker PECAM (green) showed that tdTomato expressing cells colonized the posterior lateral plate and intermediate mesoderm (yellow arrows in C″), the hindgut endoderm (red arrow in (C″)), the notochordal plate (white arrow in C″) and the endothelial lining of the dorsal aortae (paired green arrows in C″) and the omphalomesenteric vein (lower green arrow in C″). DA (dorsal aortae); HG (hindgut); ISV (inter-somitic vessels); LPM (lateral plate mesoderm); NP (notochordal plate); OV (omphalomesenteric vein).
	Fig. 6. Wnt11-CreER expression terminates in the embryonic endoderm and the extra-embryonic vasculature shortly after gastrulation. (A–D) In embryos in which a single dose of tamoxifen was administered at E7.5, the contribution of tdTomato expressing cells to the mid- (B) and hindgut (C) endoderm was reduced, and could no longer be found in the foregut endoderm (A and D). In contrast, abundant contribution of tdTomato expressing cells could be found in the splanchnic mesenchyme (SM) surrounding the hindgut (C) and in the vasculature. (E–L) When tamoxifen was administered at E8.5–9.0, tdTomato expressing cells were completely absent from the foregut (white arrows in E and H), midgut (white arrows in F) and hindgut (white arrow in G) endoderm, but contributed specifically to the nephric duct (ND in G). The contribution of tdTomato expressing cells to the extra-embryonic vasculature in the yolk sac and placenta was also diminished. (I) Whole mount epi-fluorescence alone, and (J) merged image with brightfield revealed no tdTomato expressing cells in the yolk sac. (K and L) Bisection of the placenta revealed that tdTomato expressing cells also could no longer be found in the fetal vasculature (compare to Fig. 4C–D″). The limited remaining tdTomato expression appeared to be in the extra-embryonic Reichert׳s membrane. ND (nephric duct); NT (neural tube); SM (splanchnic mesenchyme).
	Fig. 7. Wnt11-CreER expression is initiated in the endocardial progenitors during gastrulation and maintained in the differentiated endocardium. (A–B′) Activation of tdTomato expression by Wnt11-CreER was induced with tamoxifen at E6.5, and embryos were analyzed at E8.5. (A) Whole mount epi-fluorescent image of tdTomato alone, and (A′), merged with brightfield. (B, B′) Transverse sectioning and co-immunostaining with PECAM revealed that tdTomato expressing cells occupied almost the entire endocardium lining all chambers of the forming heart tube (outlined by dotted white line), including the inflow tract (IFT), the left ventricle and the right ventricle/OFT. By contrast, only few tdTomato expressing cells could be found in the LV myocardium (white arrows in B and B′). (C, C′) Tamoxifen induction at E7.5 revealed that tdTomato expressing cells contributed exclusively to the endocardium (green arrows in C and C′), but not the myocardium, in the OFT and RV. (D–F′) When tamoxifen induction was performed at E10.5 and the resulting embryos analyzed at E12.5, tdTomato expressing cells could still contribute to the entire endocardium of both the right and the left ventricles, indicating that Wnt11-CreER expression was maintained in the differentiated endocardium. In these embryos, tdTomato expressing cells also contributed to the epicardial layer around the ventricles (yellow arrows in E, E′, F and F′). en (endocardium); epi (epicardium); IFT (inflow tract); LV (left ventricle); myo (myocardium); OFT (outflow tract); RV (right ventricle).
	Fig. 8. Sequential activation of dynamic Wnt11-CreER expression in the progenitors of the ventricular myocardium. (A–D) When tamoxifen was administered at E7.0 (A, B) and 7.5 (C, D), tdTomato-labeled cells contributed extensively to the myocardium (MF20 positive, green) in the left ventricle and the inter-ventricular septum, but were absent from the myocardium of the right ventricle and OFT. (E–H) When tamoxifen was administered at E8.0, tdTomato-positive cells contributed to both the left and the right ventricular myocardium, but not to the atrial or OFT myocardium (F, H). (I–L) On the hand, when tamoxifen was administered at E8.5, tdTomato labeled cells contributed to the right ventricular myocardium (K), but their contribution to the left ventricular myocardium became diminished (L). (M–P) Finally, when tamoxifen was administered at E9.5, the contribution of tdTomato-positive cells to the left (P) and the right (O) ventricular myocardium was diminished. In all the stages analyzed above, tdTomato positive cells contributed extensively to the endocardium in all chambers. Additionally, sporadic tdTomato expressing cells were present in the epicardium of embryos that were tamoxifen-induced at E8.5 (yellow arrow in J). The entire epicardium became tdTomato positive when tamoxifen was administered at E9.5 (yellow arrows in M–P). Insets in K, L, O and P show a magnified view of tdTomato expressing cells and MF20 positive myocardium. en (endocardium); epi (epicardium); IFT (Inflow tract); LV (left ventricle); myo (myocardium); OFT(outflow tract); RV (right ventricle).
	Fig. 9. Spatially restricted contribution of Wnt11-CreER expressing cells to the OFT myocardium. (A–C) Whole mount epi-fluorescent analysis showed that when tamoxifen was administered at E8.5; in the resulting E9.5 embryos, tdTomato expressing cells were largely present in the mesoderm of the second (2nd) pharyngeal arch (black box in A and magnified view in B), the myocardium of the OFT superior wall and the right ventricle (blue box in A and magnified view in C), and the endocardium of the OFT, RV and IFT. (D–I) When embryos were harvested at E10.5 after tamoxifen induction at E9.0, tdTomato expressing cells were observed along the proximo-distal axis of the superior myocardial wall of the OFT (white arrow in D and F), which also co-expressed myocardial marker MF20. Transverse section of the OFT (G–I) indicated that the tdTomato expressing cells occupied ~3/4 of the superior and lateral OFT myocardial wall and were missing from the inferior OFT wall (white dotted line in G–I). When E9.0 tamoxifen induced embryos were harvested at E12.5, transverse sectioning and co-staining with MF-20 indicated that tdTomato labeled cells were present specifically in the sub-aortic myocardium (white arrows in J, L, M and O), but were absent from the sub-pulmonary myocardium (white dotted lines in J–O). In contrast, co-staining with PECAM indicated that tdTomato labeled cells contributed to the endocardium/endothelium in both the aorta and pulmonary artery. Ao (aorta), BA (branchial arch); en (endocadium); IFT (inflow tract); OFT (outflow tract); PA (pulmonary artery); PhA (pharyngeal arch); RV (right ventricle).

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