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Fig. S1. Allantois growth and expression of a4 integrin and Vcam1. (A) The length of the non-fused Cdx4+/– (in yellow), Cdx2+/–/Cdx4+/– (in red) and Cdx2+/–/Cdx4–/0 (in blue) allantois was measured and compared with the somite number of the embryo. Although the length of the allantois at a given somite stage appears to be variable, the measurements do not suggest an obvious length reduction in Cdx2+/–/Cdx4–/0 mutant allantois compared with the Cdx4+/– control allantois. (B-E) The mesodermal layer of the chorion is present in chorio-allantoic fusion defective embryos and expresses a4 integrin (Yang et al., 1995). (B,C) Histological analysis of E9.5 wild-type and Cdx2+/–/Cdx4–/0 mutant placentas. (B) After chorio-allantoic fusion in wild-type placentas, allantoic mesenchyme is attached to the trophoblast. The chorionic mesodermal layer is absent as the fusion occurs at the expense of the chorionic mesoderm (Downs, 2002). (C) The Cdx2+/–/Cdx4–/0 compound mutant placenta that had failed to undergo chorio-allantoic fusion is devoid of allantoic mesenchyme, and the mesodermal layer of the chorion is visible (arrow in C). (D,E) Expression of a4 integrin in E8.5 wild-type and Cdx2+/–/Cdx4–/0 mutant placentas. (D) The wild-type placenta does not express a4 integrin anymore as its chorionic mesoderm is broken down. (E) Cdx2+/–/Cdx4–/0 placentas that are defective in chorio-allantoic fusion, express a4 integrin in the mesodermal layer of the chorion (arrowhead). The loose attachment of the mesodermal compartment is an artefact of sectioning. (F,G) Expression of Vcam1 in the allantois. (F) Vcam1 is expressed in the wild-type allantois (eight somites) at the time of chorio-allantoic fusion (Gurtner et al., 1995). (G) Vcam1 expression is also detected in the Cdx2+/–/Cdx4–/0 mutant allantois (nine somites) that failed to fuse with the chorion. CE, chorionic ectoderm; CM, chorionic mesoderm; AM, allantoic mesenchyme; Tr, trophoblast. Scale bars: 50 mm in B and C; 200 mm in D and E; 300 mm in F,G.
References
Downs, K. M. (2002). Early placental ontogeny in the mouse. Placenta 23, 116-131.
Gurtner, G. C., Davis, V., Li, H., McCoy, M. J., Sharpe, A. and Cybulsky, M. I. (1995). Targeted disruption of the murine VCAM1 gene: essential role of VCAM-1 in chorioallantoic fusion and placentation. Genes Dev. 9, 1-14.
Yang, J. T., Rayburn, H. and Hynes, R. O. (1995). Cell adhesion events mediated by alpha 4 integrins are essential in placental and cardiac development. Development 121, 549-560.
Fig. S2. Analysis of gene expression from wild-type and Cdx2+/–/Cdx4–/0 in E8.25 individual allantoises before chorio-allantoic fusion. (A) RT-PCR shows a panel of genes known to play important roles in vascular development (Tie1 and Tie2, Flk1 and Flt1, Pecam, Vegf) and chorio-allantoic fusion (Vcam1). a-Actin is shown as an internal control. No obvious expression differences were noted between wild-type and mutant allantois. (B) RT-PCR analysis of gene expression in E9.25 wild-type and Cdx2+/–/Cdx4–/0 labyrinths shows slightly lower expression of Gcm1, which is known to be involved in penetration of the allantoic vessels into the chorionic ectoderm. Other panels show expression of Agpt, Agpt2 and Vegf, genes known to be involved in angiogenesis; the expression of these genes is not conclusively changed in the mutants. a-Actin is shown as internal control. RNA isolation and RT-PCR. Total RNA was isolated from wild-type and Cdx2+/–/Cdx4–/0 individual allantoises of five- to seven-somite (E8.25) embryos using Qiagen micro RNA isolation kit (Qiagen). First-strand cDNA was synthesized from RNA (1 mg) using a SuperScript Preamplification System kit (Gibco-BRL). A fivefold dilution of first strand cDNA was used for semi-quantitative RT-PCR with specific published oligonucleotides for Tie1 (Vittet et al., 1996) (30 cycles), Tie2 (Vittet et al., 1996) (32 cycles), Flk1 (Vittet et al., 1996) (27 cycles), Flt1 (Feraud et al., 2001) (32 cycles), Pecam (Vittet et al., 1996) (30 cycles), Vcam1 (Cybulsky et al., 2001) (35 cycles), Agpt (Yang et al., 2000) (32 cycles), Agpt2 (Yang et al., 2000) (30 cycles), Gcm1 (Maret et al., 2004) (26 cycles) and a-actin (Yang et al., 2000) (25 cycles). PCR products were separated on a 1.5% agarose gel and visualized by ethidium bromide staining.
References
Cybulsky, M. I., Iiyama, K., Li, H., Zhu, S., Chen, M., Iiyama, M., Davis, V., Gutierrez-Ramos, J. C., Connelly, P. W. and Milstone, D. S. (2001). A major role for VCAM-1, but not ICAM-1, in early atherosclerosis. J. Clin. Invest. 107, 1255-1262.
Feraud, O., Cao, Y. and Vittet, D. (2001). Embryonic stem cell-derived embryoid bodies development in collagen gels recapitulates sprouting angiogenesis. Lab. Invest. 81, 1669-1681.
Maret, A., Bourdeau, I., Ding, C., Kadkol, S. S., Westra, W. H. and Levine, M. A. (2004). Expression of GCMB by intrathymic parathyroid hormone-secreting adenomas indicates their parathyroid cell origin. J. Clin. Endocrinol. Metab. 89, 8-12.
Vittet, D., Prandini, M. H., Berthier, R., Schweitzer, A., Martin-Sisteron, H., Uzan, G. and Dejana, E. (1996). Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps. Blood 88, 3424-3431.
Yang, J., Boerm, M., McCarty, M., Bucana, C., Fidler, I. J., Zhuang, Y. and Su, B. (2000). Mekk3 is essential for early embryonic cardiovascular development. Nat. Genet. 24, 309-313.
Fig. S3. Assay of apoptosis and proliferation in Cdx2+/–/Cdx4–/0 and control labyrinths. (A-D) Assay of apoptosis in Cdx mutant and controls at E9.5 and E10.5. (A,B) Immunohistochemical analysis of anti-cleaved caspase 3 staining in E9.5 wild-type and Cdx2+/–/Cdx4–/0 compound mutant placentas reveals an absence of apoptotic cells in the chorio-allantoic region of the placenta, and comparable numbers of apoptotic cells in the ectoplacental cone (C,D). Apoptotic cells are seen in higher numbers at E10.5 in endothelial cells, but also in blood cells from the compound mutant embryo (arrowheads in D, compare with C), suggesting that it foreshadows the approach of embryonic death. (E,F) Typical field crossing endothelial vessels in sections of Cdx2+/–/Cdx4–/0 and control E9.5 labyrinths immunoassayed with an anti-Ki67 antibody, showing that all endothelial cells clearly identifiable are in a proliferation phase of the cell cycle in mutant and control (arrowheads in E,F). Triangles in E and F indicate nucleated embryonic red blood cells. Scale bars: 200 mm in A-D; 30 mm in E,F. cp, chorionic plate; epc, ectoplacental cone; mbs, maternal blood sinus; frb, fetal red blood cell; la, labyrinthine trophoblast.
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