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First published online 5 January 2006
doi: 10.1242/dev.02216

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The Cdx4 mutation affects axial development and reveals an essential role of Cdx genes in the ontogenesis of the placental labyrinth in mice

¹ Hubrecht laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8 3584 CT Utrecht, The Netherlands.
² Faculté de Médecine Pitié - Salpêtrière, 91 Boulevard de l'Hôpital UMRS 525 INSERM/UPMC, 75634 Paris, Cedex 13, France.
³ University of Leicester, Department of Biochemistry, University Road, Leicester LE1 7RH, UK.

View larger version (71K): [in a new window]   Fig. 1. Targeted disruption of the Cdx4 gene. (A) The gene-targeting vector inserts an antisense floxed PGK-hygror cassette into an EcoRV site upstream of exon 2 and an antisense flipped PGK-neor cassette, including a single loxP site into an EcoRI site downstream of exon 3, resulting in a conditional allele of Cdx4 (Cdx4c). Cre-mediated deletion of the homeodomain in exon 2 and exon 3 generates the Cdx4-null allele (Cdx4-). LoxP and FRT sites are represented by closed and open red triangles, respectively. HIII, HindIII; KI, KpnI; RI, EcoRI; RV, EcoRV; SI, SpeI. (B) Southern blot analysis of male ES cell clones with a unique 5' genomic probe reveals a 17 kb KpnI fragment generated by the targeted conditional allele. A 3' internal probe reveals correct 3' targeting by identification of a 17 kb KpnI fragment. (C) Histological evaluation of the digestive tract of adult Cdx4-null and control (wild-type) mice. Ki67 immunohistochemistry shows normal proliferation in the intestinal and colonic crypts (brown cells). Unlike previous findings in Cdx2+/- mice, no intestinal polyp-like lesions were observed.

View larger version (73K): [in a new window]   Fig. 2. Phenotype of compound Cdx2+/-/Cdx4-/0 mutant embryos. (A) Gross morphology of a wild-type and Cdx2+/-/Cdx4-/0 compound mutant littermate at E10.5, showing developmental retardation of the compound mutant embryo. Thoracic edema (arrowhead in A) indicates a defect in embryonic circulation. (B,C) Close up of the tail bud of a E10.25 wild type (B) and Cdx2+/-/Cdx4-/0 mutant (C), showing the posterior truncation. TB, tail bud; HL, hindlimbs; FL, forelimbs. Scale bar: 500 µm.

View larger version (61K): [in a new window]   Fig. 3. A subset of Cdx2+/-/Cdx4-/0 mutant embryos fails to undergo chorio-allantoic fusion. (A) Lateral view of freshly dissected E9.5 wild-type embryo. The allantois has fused to the chorionic part of the placenta (arrow). (B) Lateral view of a E9.5 Cdx2+/-/Cdx4-/0 compound mutant littermate that is defective in chorio-allantoic fusion (arrow). (C) Frontal view of the embryo shown in B. The allantois appears to end as a massive ball of cells (arrow). (D) Histological analysis of the allantois shown in C. Several allantoic vessels, containing nucleated embryonic blood cells (arrowheads) and the outer mesothelial layer (arrow) are visible. Pl, placenta; TB, tail bud. Scale bar: 100 µm.

View larger version (101K): [in a new window]   Fig. 4. Defective placental labyrinth development in Cdx2+/-/Cdx4-/0 compound mutant embryos. (A-H) Hematoxylin and Eosin-stained sections of placentas from wild-type (A,C,E,G; C is an enlargement of A; G is an enlargement of E) and Cdx2+/-/Cdx4-/0 compound mutant littermates (B,D,F,H; D is an enlargement of B; H is an enlargement of F). (A-D) At E9.5, only some Cdx2+/-/Cdx4-/0 allantoic vessels have started to penetrate the chorionic trophoblast layer without overt signs of branching morphogenesis, whereas wild-type placentas show branched vessels deeply penetrating the chorionic ectoderm. These vessels intermingle with maternal blood sinuses, from which they are separated in many places by only a thin haemotrichorial membrane (triangle in C) in the crucial part of the labyrinth (la in A) that is concerned with the vital interchanges between maternal and fetal circulation. (E-H) At E10.5, the defect becomes more severe, as revealed by the complete separation of maternal and embryonic blood flows (mrb and frb in H) and the virtual absence of the labyrinth (F, compare with E). Scale bars: 100 µm. cp, chorionic plate; mrb, maternal red blood cell; frb, fetal red blood cell; la, labyrinthine trophoblast.

View larger version (64K): [in a new window]   Fig. 5. Altered branching of allantoic vessels, and similar endothelial and trophoblast marker expression in Cdx mutant and control embryos. (A,B) Morphometric analysis of embryonic placental vessels reveals more vessels with smaller average diameter in wild-type placentas than in mutant placentas. (C,D) Immunohistochemical staining for Pecam marks endothelial cells (in brown) in the developing labyrinth. At E9.5, wild-type embryonic vessels have penetrated the chorionic ectoderm and branched extensively (arrowheads in C) while Cdx2+/-/Cdx4-/0 mutant endothelial cells can only be detected on one side of the chorionic plate (arrowhead in D). (E,F) A well-established labyrinth is present at E10.5 in wild types (la in E), whereas in Cdx2+/-/Cdx4-/0 mutant placentas, embryonic vessels are present only in the allantoic mesoderm (F). (G-N) Expression of trophoblast markers at E9.5 in Cdx2+/-/Cdx4-/0 compound mutant placentas and controls. (G,H) In situ hybridization of Cdx2 shows expression in the ectoplacental cone and spongiotrophoblast. Expression is absent in the labyrinthine trophoblasts in both mutants and controls. (I,J) In situ hybridization of Mash2 shows expression in the ectoplacental cone, spongiotrophoblasts and labyrinthine trophoblasts. The morphology of the labyrinthine trophoblast differs between mutant and control, owing to the absence of an extensively intermingled labyrinth in the compound mutant. (K,L) In situ hybridization of Hand1 (eHAND) shows expression in the spongiotrophoblast and labyrinthine trophoblasts in wild-type and compound mutant placentas. (M,N) The ectoplacental cone and spongiotrophoblast marker Tpbp is expressed in wild-type and Cdx2+/-/Cdx4-/0 compound mutant placentas. epc, ectoplacental cone; sp, spongiotrophoblasts; tr, labyrinthine trophoblasts; cp, chorionic plate; la, placental labyrinth; mbs, maternal blood sinus; fbv, fetal blood vessel; Cdx mutant, Cdx2+/-/Cdx4-/0. Scale bars: 100 µm C-F; 500 µm in G-N.

View larger version (73K): [in a new window]   Fig. 6. Cdx expression and early vascular development in the allantois. (A-C) In situ hybridization on E7.5 embryo sections reveals strong expression of Cdx1 in the primitive streak and weak expression at the base of the allantois (arrowhead in A). Expression is absent in the chorion (asterisk in A). (B) Cdx2 is strongly expressed in the primitive streak and in the outgrowing allantois (arrowhead) and in the chorion (triangle). (C) Cdx4 expression is restricted to the primitive streak and allantois (arrowhead), and is absent in the chorion (asterisk). (D,E) Flk1 staining marks endothelial precursors that are present throughout the E7.5 wild-type (D) and Cdx2+/-/Cdx4-/0 mutant allantois (E). The allantois of the wild type was slightly smaller than that of the compound mutant, explaining the lower Flk1 staining in D. (F-I) Pecam staining of allantoises isolated at E8.25 just after chorio-allantoic fusion. Primary endothelial networks in wild-type (F and H) and Cdx2+/-/Cdx4-/0 mutant (G and I) allantoises are visible. The wild-type E8.25 allantois in F shows organization of endothelial cells into vessels with numerous branching and connecting vessels (arrowheads). Endothelial cells are less well organized in Cdx2+/-/Cdx4-/0 compound mutant littermate shown in G, with clumps of Pecam-positive cells (arrows). The Cdx2+/-/Cdx4-/0 allantois in I is, in this case, smaller than the wild-type control (H). Scale bars: 200 µm in A-C; 40 µm in D,E; 20 µm in F,G; 80 µm in H,I. D, distal; P, proximal.