Placental failure in mice lacking the homeobox gene Dlx3 - PubMed (original) (raw)

Placental failure in mice lacking the homeobox gene Dlx3

M I Morasso et al. Proc Natl Acad Sci U S A. 1999.

Abstract

Dlx3 is a homeodomain transcription factor and a member of the vertebrate Distal-less family. Targeted deletion of the mouse Dlx3 gene results in embryonic death between day 9.5 and day 10 because of placental defects that alter the development of the labyrinthine layer. In situ hybridization reveals that the Dlx3 gene is initially expressed in ectoplacental cone cells and chorionic plate, and later in the labyrinthine trophoblast of the chorioallantoic placenta, where major defects are observed in the Dlx3 -/- embryos. The expression of structural genes, such as 4311 and PL-1, which were used as markers to follow the fate of different derivatives of the placenta, was not affected in the Dlx3-null embryos. However, by day 10.5 of development, expression of the paired-like homeodomain gene Esx1 was strongly down-regulated in affected placenta tissue, suggesting that Dlx3 is required for the maintenance of Esx1 expression, normal placental morphogenesis, and embryonic survival.

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Figures

Figure 1

Figure 1

Targeted disruption of Dlx3. (A) Restriction map of the Dlx3 gene, the targeting vector, and the predicted restriction map after the homologous recombination event. The probes used and the predicted length of restriction fragments in the Southern blot analysis are shown. B, _Bam_HI; K, _Kpn_I; E, _Eco_RI; S, _Sma_I; X, _Xba_I; NEO, neomycin phosphotransferase gene; TK, thymidine kinase gene. (B) Genomic analysis of wild-type-derived ES cell DNA (lanes I) and two independently targeted ES cell clones (lanes II and III). Recombination was detected at the 5′ end by digesting ES cell DNA with _Kpn_I/_Asp_718 and hybridizing the Southern blot with 5′ probe, and at the 3′ end by digesting DNA with _Eco_RI and _Sma_I and hybridizing with 3′ probe. A single integration event was confirmed by hybridizing _Kpn_I/_Asp_718-digested DNA with probe Neo (Center). Numbers at sides of the figure indicate fragment size in kb. (C) Genotyping of tail-derived DNA revealing the expected pattern for wild-type (wt), heterozygous (+/−), and homozygous _Dlx3-_null (−/−) embryonic day 9.5 (E9.5) embryos. (D) Phenotype of wild-type (left) and _Dlx3-_null (right) E9.5 embryos genotyped by Southern blotting. (E) Phenotype of wild-type (left) and regressed Dlx3 −/− (right) E12.5 embryos genotyped by Southern blotting.

Figure 2

Figure 2

Expression of Dlx3. In situ hybridization with antisense Dlx3 of sagittal sections of wild-type conceptus within the uterus at E8.5 (A; scale bar = 400 μm) and placentas from wild-type embryos at 9.5 (B; scale bar = 300 μm) and 10.5 (C; scale bar = 500 μm) days of development. (D) Northern blot of 10 μg each of RNA from E16.5 placenta (P), neonatal epidermis (E), and adult brain (B) hybridized with a Dlx3 cDNA probe. (Upper) Hybridization with Dlx3 probe; number at the right indicates RNA size. (Lower) Ethidium bromide staining of 18S ribosomal RNA.

Figure 3

Figure 3

(A) Trophoblast tissues from Dlx3 +/+ (Left) and −/− (Right) littermates. Histology of sagittal sections of +/+ and −/− placentas at E9.5, E10.5, and E12.5 stained with hematoxylin and eosin. al, allantois; la, labyrinthine layer; sp, spongiotrophoblast layer, gi, giant trophoblast layer; d, decidua. (Scale bar = 250 μm.) (B) Higher magnification of boxed areas in A for panels +/+ and −/− at E12.5. Arrowhead indicates maternal enucleated blood cell and arrow indicates fetal blood cell. A few blood vessels are apparent in the compacted labyrinthine and spongiotrophoblast layers in the −/− E12.5 placenta. (Scale bar = 40 μm.)

Figure 4

Figure 4

Expression of cell-type-specific markers analyzed by in situ hybridization of sagittal sections of conceptus at E9.5, and placentas from Dlx3 +/+ and −/− embryos at E10.5. Hybridizations were performed with antisense probes for 4311 (spongiotrophoblast cells and their precursors in the ectoplacental cone); PL-1 (trophoblast giant cell); mGCNF (labyrinthine layer); Mash-2 (spongiotrophoblast and labyrinthine layers and chorion); and Esx1 (endoderm layer of the visceral yolk sac and labyrinthine trophoblast of the chorioallantoic placenta). (×10.) At the bottom of the figure there is a schematic representation of E10.5 placenta indicating the different cell types.

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