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First published online 17 January 2006
doi: 10.1242/jcs.02769

Journal of Cell Science 119, 482-489 (2006)
Published by The Company of Biologists 2006
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Desmocollin 3 is required for pre-implantation development of the mouse embryo

Zhining Den1,*, Xing Cheng1,*, Maria Merched-Sauvage1 and Peter J. Koch1,2,{ddagger}

1 Department of Dermatology, Baylor College of Medicine, Houston, TX 77030, USA
2 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA


Figure 1
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  		Fig. 1. Schematic representation of the targeting strategy used to generate Dsc3+/– ES cells. The map is not drawn to scale. (A) The 5' end of the Dsc3 gene is shown. Exons are represented by vertical boxes. The location of the start codon (ATG) is indicated. Horizontal bars indicate the positions of the external probes used to identify targeted ES cells in Southern blots. (B) Structure of the Dsc3 gene targeting vector. A fragment containing promoter elements, exon 1 and part of intron 1 was deleted and replaced by a neomycin-resistance mini gene (PGK-Neo) which was flanked by loxP sites (triangles). (C) Structure of the recombinant Dsc3 locus after homologous recombination. (D) Dsc3-null allele after excision of the PGK-Neo cassette via Cre-mediated recombination (see text for details). (E) EcoRV-digested genomic DNA from Dsc3 line 1 mice was tested with the external probes. The 5' probe detected a 13.5 kb wild-type fragment and a 4.3 kb mutant fragment in Dsc3+/– DNA. The fragment sizes for the 3' probe were 13.5 kb (wild type) and 6.7 kb (mutant). The neo probe detected a single band of 6.7 kb. (F) Western blot analysis of ear lysates from wild-type (+/+) and adult Dsc3+/– (+/–) mice using antibodies against Dsc3, plakoglobin (Pg) and ß-catenin. A quantitative analysis of the Dsc3 signals (using the ß-catenin signal as a loading control) revealed that Dsc3 synthesis was reduced by half in the Dsc3+/– sample. This was particularly obvious when comparing the intensities of the Dsc3b bands in wild-type and mutant mice. (G) QPCR analysis of Dsc3 mRNA levels in wild-type (WT) and adult Dsc3+/– (Het) ear. Two pairs of heterozygous mutants and wild-type littermates were used in this analysis. Dsc3 expression was approximately halved in ear.

 

Figure 2
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  		Fig. 2. Dsc3 expression throughout mouse development. (A) A northern blot (Seegene, Seoul, Korea) with total RNA from various stages of development was hybridized to a Dsc3 exons 4-8 probe. We obtained only a weak signal at E11.5. (B) RT-PCR analysis with RNA samples from E7.5-E13.5 embryos. RNA from adult mouse skin and kidney served as positive and negative controls, respectively. Band intensity does not necessarily correspond to expression levels in these experiments. (C) RT-PCR using cDNA from E3.5 embryos. Dsc2, Dsc3 and Dsg2 are expressed. Epidermal RNA served as a positive control. (D) Western blot with our Dsc3 antibody, demonstrating that Dsc3a and Dsc3b are both present in total cell lysates from wild-type blastocyst-stage embryos. The blot was stripped and re-probed with ß-catenin antibodies as a control. (E) RT-PCR analysis of E2.5 embryos (pre-compaction stage eight-cell embryos). Two desmosomal cadherins are expressed at this stage: Dsc3 and Dsg2. We also detected mRNA for plakoglobin (PG) and desmoplakin (DP). ß-Actin primers were used as a positive control. (F) RT-PCR with RNA from unfertilized oocytes demonstrating the presence of Dsc3 mRNA. Mouse epidermal RNA served as a positive control. ß-Actin primers were used to confirm the presence of intact cDNA. Blank indicates a negative control, no RNA added.

 

Figure 3
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  		Fig. 3. Detection of Dsc3 in pre-implantation embryos. (A) Immunofluorescence staining of blastocysts with antibodies against Dsc3, desmoplakin (Dp) and ß-catenin (ß-Cat). The two blastocysts shown were co-stained either with Dsc3 (red) + DP (green) or with Dsc3 (red) + ß-cat (green) antibodies. Co-localization of the antigens is indicated by yellow fluorescence. The boxed areas are shown at higher magnification on the right. Dsc3 is distributed in a linear fashion along the plasma membrane of TE cells, i.e. does not show the typical punctuated desmosomal staining pattern. Bars, 20 µm. (B) Immunohistochemistry on wild-type pre-implantation stage embryos and ovary sections using our Dsc3 antibody. All embryos showed a homogenous cytoplasmic staining. The Dsc3 antibody stains the cytoplasm of oocytes in ovary sections. The box marks an area that is shown at higher magnification on the right. Control sections incubated with normal guinea pig antibodies (nIgG) showed only background staining. Arrows indicate polar bodies in the two-cell stage embryos. N, nucleus; Cy, cytoplasm.

 

Figure 4
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  		Fig. 4. In vitro development of pre-implantation embryos from Dsc3+/– (line 1) intercrosses and the PCR strategy used for genotyping. Embryos were isolated at E1.5 and then cultured for up to 4 days. The pictures on the upper row were taken 2 days after embryo isolation (pre-compaction eight-cell stage; E2.5 equivalent). (A) The different stages of development as seen in the wild-type embryo are indicated on the left (morula, early blastocyst, blastocyst with expanded blastocoel cavity). A wild-type embryo (+/+), a heterozygous mutant (+/–) and four homozygous mutants (–/–) are shown. Heterozygous mutants were morphologically normal. Most mutant embryos, consisting of multiple blastomeres, were disintegrated at the time when normal embryos undergo compaction. (B) Three primers (I, II, III) were designed to distinguish the Dsc3 wild-type (WT) and mutant (Mut) allele. These primers allow identification of all three genotypes (wild type, heterozygous and homozygous mutants) in a single PCR reaction. The PCR products were hybridized to primer H in order to confirm that they were derived from the Dsc3 gene locus (see autoradiogram on the right).

 

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