Fig. 6. LPP3 deficiency results in axis duplication. (A,B) An E9.5 LPP3^-/-conceptus where the anterior part (hf) of the embryo developed outside the yolk sac (ys) and also exhibited abnormal vasculogenesis (A). When dissected from the extra-embryonic membranes (B), the embryo showed abnormal development of the allantois (all). (C) Cross section through an abnormal embryo showing deficiency in mesenchymal tissue around the paraxial mesoderm. Two large blood vessels (bv) have developed ventral to the somites. d, dorsal. (D) Higher magnification of C showing the neural tube (nt) clearly duplicated in the ventral region, in which a double notochord (^*) is also evident. A third somite (s) row has formed ventrally to both notochords. (E,F) Shh and Twist double whole-mount in situ hybridization in LPP3^-/- embryos with axis duplication. (E) In some embryos, two short Shh-positive notochords (n) were observed without an additional somite row forming between them. (F) In others, a clear Twist-positive extra somite row formed between the duplicated Shh-positive notochords. (G-H) At E8.5 (G) and E7.5 (H) LPP3^-/- embryos (right) develop outside the yolk sac unlike the nornal littermate (left). A constriction between the embryonic and extra-embryonic tissues (arrowhead) is present. In the null embryos the brachyury-expressing primitive streak is shorter in the mutant embryo that in the wild type. A, anterior; P, posterior. (G) E8.5 embryos, (H) E7.5 embryos. (I) On the left, two E7.0 embryos show normal brachyury expression in the primitive streak. On the right, two abnormal littermates show retarded development (equivalent to E6.5), one of which shows primitive streak duplication (arrows). (J) Posterior view of a LPP3^-/- embryo recovered at E8.5. Brachyury expression revealed the presence of a common primitive streak-like structure from which two axial mesoderm-like structures have formed. (K) dkk1 expression in an E7.5 LPP3 heterozygous (left) and homozygous mutant (right) embryos. While dkk1 expression extends from the proximal AVE to the distal visceral endoderm in the heterozygous embryo, in the LPP3 null embryo weak dkk1 staining is restricted to a band of cells located in the distal visceral endoderm (arrow). An abnormal outgrowth of tissue formed exactly below the dkk1 expression domain of this mutant embryo (small arrow). The arrowheads indicate the junction between the embryonic and extra-embyonic tissues. A, anterior; P, posterior. (L) Hex and Wnt3 expression are also altered in E7.0 LPP3 null embryos. Hex-positive cells accumulate in the distal tip of the egg cylinder and Wnt3 expression is also found in the anterior embryonic ectoderm (arrows). In normal embryos Hex is expressed in the AVE and Wnt3 is restricted to the posterior embryonic ectoderm. Bars: 250 µm.

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