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First published online 30 November 2006
doi: 10.1242/dev.02722

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Gene replacement reveals a specific role for E-cadherin in the formation of a functional trophectoderm

Natalia G. Kan^1,*, Marc P. Stemmler¹, Dirk Junghans¹, Benoît Kanzler¹, Wilhelmine N. de Vries², Mara Dominis³ and Rolf Kemler^1,

¹ Max-Planck-Institut für Immunbiologie, Abteilung für Molekulare Embryologie, Stübeweg 51, D-79108 Freiburg, Germany.
² The Jackson Laboratory, Bar Harbor, ME 04609-1500, USA.
³ Department of Pathology, Clinical Hospital Merkur, 4100 Zagreb, Croatia.

View larger version (70K): [in this window] [in a new window]   Fig. 1. Generation of Ncad k.i. mice. (A) Targeting strategy with schematic representation of the 5' region of the Ecad genomic locus including promoter (P) and exons 1 and 2 (E1 and E2). Targeting vectors and expected recombined alleles are shown. Sites for restriction endonucleases and probes used are indicated. The neomycin selection cassette is flanked by two loxP sites (red triangles). (B) Southern blot analysis of targeted ES cell clones. DNA was digested with XbaI and probed with a 5' probe (upper panel) located upstream of the targeting vectors (see also Fig. 1A). Fragments of 6.6 kb and either 7.8 kb or 8.5 kb were detected, corresponding to wt and Ncad k.i. or Ncad-GFP k.i. alleles, respectively. The internal probe was located downstream of exon 2. Fragments of 8.8 kb and either 9.3 kb or 10.1 kb, corresponding to wt and Ncad k.i. or Ncad-GFP k.i. alleles, respectively, were detected with SpeI-digested DNA (lower panel). Corresponding genotypes are indicated. (C) Double immunofluorescence against E- and N-cad on wt and targeted ES cell clones. N-cad is expressed from the k.i. allele and co-localizes with E-cad at the cell membrane. Wild-type ES cells are negative for N-cad immunoreactivity. (D) RT-PCR analysis of Ncad-GFP k.i. expression in different organs at E15.5. cDNAs from indicated organs of wt (+/+) or Ecad+/Ncad (+/k.i.) embryos were used with specific primers to amplify transcripts of Ecad, endogenous Ncad and Ncad-GFP. (E) Immunohistochemistry of wt and heterozygous Ncad k.i. embryos at E15.5. E-cad shows membrane localization in the intestine epithelium of wt and heterozygous Ncad k.i. embryos. N-cad k.i. protein is specifically detected in the intestinal epithelium of embryos carrying one Ncad k.i. allele but not in wt embryos (arrows). Endogenous N-cad protein is detected in peripheral muscle cells of the intestine in both wt and heterozygous embryos (arrowheads). Scale bar: 50 µm.

View larger version (35K): [in this window] [in a new window]   Fig. 2. Homozygous Ncad k.i. mutants fail to form an intact trophectoderm. (A-B'') Double immunolabeling of E- and N-cad protein. E-cad is localized at the cell-cell contact sites in wt (A) and heterozygous Ncad k.i. E4.5 blastocysts (A'), but is absent in homozygous Ncad k.i. embryos (A''). N-cad immunolabeling is observed in heterozygous (B') and homozygous mutants (B''). No N-cad staining above background is observed in wt embryos (B). Co-localization of E- and N-cad at cell-cell contact sites of both ICM and TE is seen in heterozygous preimplantation embryos (A',B'). Additional punctate staining of N-cad is seen on the apical membrane of TE cells (B', arrow). (C-E''') Time-lapse analysis of the preimplantation development of Ncad k.i. embryos. Recordings were started at the 8-cell stage (C-C'') and continued for 24 hours. Representative pictures of recordings are shown. At the 8-cell stage, wt (C), heterozygous (C') and homozygous (C'') embryos were indistinguishable, and all developed normally to compacted morulae (D-D''). (E-E''') Representative pictures of in vitro-cultured E4.5 embryos. Wt (E) and Ecad+/Ncad (E') embryos formed expanded blastocysts, whereas homozygous EcadNcad/Ncad mutants showed loosening of cell-cell contacts and formed either no cavity (E'') or only small cavity-like cysts (E'''). (F) RT-PCR analysis of single E3.5 blastocysts: wt (1), heterozygous (2) and homozygous k.i. (3) embryos with primers for Ecad and Ncad mRNA as indicated. Scale bar: 25 µm.

View larger version (38K): [in this window] [in a new window]   Fig. 3. Expression of trophectodermal and lineage specification markers in Ncad k.i. mutants. (A,A') Double immunolabeling of E3.5 embryos with antibodies against Oct4 (green) and Cdx2 (red) shows predominantly ICM localization of Oct4 and trophectodermal expression of Cdx2 in wt (A) and in mutant embryos (A'). Some outer cells are also Oct4-positive in mutant embryos (A'). (B,B') Monoclonal TROMA-1 antibody detects intermediate filament marker cytokeratin 8 in mutant (B') and wt (B) embryos. (C,C') Immunolocalization of ZO-1. In wt embryos ZO-1 localization is restricted to tight junctions (C). More extended and less punctate membrane localization of ZO-1 protein is seen in homozygous EcadNcad-GFP/Ncad-GFP mutants (C'). (D,D') Polyclonal anti-ezrin antibodies were used to detect microvilli on the apical pole of the outer cells in compacted wt (D) and mutant (D') embryos. (E,E') Actin cytoskeleton was detected with Alexa-488-conjugated Phalloidin. Scale bar: 25 µm.

View larger version (57K): [in this window] [in a new window]   Fig. 4. Ncad k.i. embryos can attach and form blastocyst outgrowth. DIC images of wt (A) and EcadNcad-GFP/Ncad-GFP (A') blastocyst outgrowths. Homozygous mutant embryos were able to attach and differentiate into trophoblast giant cells (arrow) and ICM (arrowhead). E-cad is expressed in the ICM and downregulated in giant cells of wt embryos (B) and is absent in the ICM of homozygous mutant embryos (B'). The trophoblast marker antibody TROMA-1 stained cells of both wt and mutant outgrowths (C-D'). D,D' are enlarged pictures of the outlined areas in C,C', respectively. Scale bar: 100 µm.

View larger version (58K): [in this window] [in a new window]   Fig. 5. Development of Ncad-GFP k.i. embryos in the absence of maternal E-cad. Representative pictures of time-lapse recordings showing embryos without maternal E-cad but expressing paternal E-cad (Ecad-/+) (A,B) or expressing N-cad-GFP from the k.i. allele (Ecad-/Ncad-GFP) (A',B'). Both embryos compact in a similar fashion around E3.0 (A,A'). By E4.0, embryos expressing paternal E-cad develop to early blastocyst (B), whereas embryos expressing paternal N-cad-GFP from the k.i. allele begin to deteriorate shortly after compaction (B'). Immunolabeling shows expression and membrane localization of E-cad from the paternal allele in Ecad-/+ embryos (C) and no E-cad expression in embryos carrying a paternal Ncad-GFP k.i. allele (C'). The same embryos were co-stained with a polyclonal anti-ezrin antibody (D,D'). Expression of N-cad-GFP was confirmed by immunostaining with a monoclonal anti-GFP antibody (E'). Only low levels of background staining are seen in embryos with a paternal E-cad allele (E). Embryos shown in E and E' were co-stained for ZO-1 (F,F'). (G-H') Oct4 and Cdx2 are expressed in Ncad-GFP k.i. embryos in the absence of maternal E-cad. 3D-reconstruction pictures from multiple optical sections show nuclear localization of Oct4 mostly in the ICM of Ecad-/+ early blastocyst (G) and inner cells of Ecad-/Ncad-GFP morulae (G'). Cdx2 is expressed exclusively in outer cells in both Ecad-/+ (H) and Ecad-/Ncad-GFP (H') embryos. Co-expression of Oct4 and Cdx2 is still observed in trophectodermal cells of E3.5 embryos. Arrows show ICM, arrowheads show TE. Scale bar: 25 µm.

View larger version (126K): [in this window] [in a new window]   Fig. 6. EcadNcad-GFP/Ncad-GFP ES cells can contribute to forming chimeric embryos. (A-C) Representative X-gal stained embryos obtained from injection of Ecad+/Ncad-GFP ES cells into ROSA26 (ROSA26;Ecad+/+) blastocysts that constitutively express lacZ. Chimeric embryos with contribution of ES cells (17 of 33 analyzed embryos showed chimerism with average ES cell contribution of 50-90%) were identified by unstained cells. (A) E8.5 chimeric embryo with approximately 50% ES cell contribution. Ecad+/Ncad-GFP ES cells can contribute to all germ layers as seen in sections at E8.5 (B). (C) E9.5 chimeric embryo with 90% ES cell-derived cells. (D-F) X-gal staining of embryos derived from injections of EcadNcad-GFP/Ncad-GFP ES cells into ROSA26 blastocysts. In 15 of 52 analyzed embryos, ES cell-derived cells are detectable, with an average contribution to the host embryo of 5-20%. Representative embryos at E8.5 with even distribution (D) and with contribution mainly to the tail at E9.5 (F, arrowhead) are shown. (E) Sections of the embryo in D revealed greater than 20% ES cell contribution to all germ layers (arrow, endoderm; black arrowhead, mesoderm; white arrowhead, ectoderm). Scale bars: A,C,F, 500 µm; B,D,E, 100 µm.

View larger version (90K): [in this window] [in a new window]   Fig. 7. ES cells derived from Ncad-GFP embryos can form epithelial structures in teratomas. (A,A') Histological stainings with hematoxylin and eosin (H&E) on paraffin sections of heterozygous (A) and homozygous (A') Ncad-GFP k.i. teratomas. Multiple epithelial-like structures are seen on both sections. (B,B') Anti-GFP immunolabeling reveals expression of N-cad-GFP in epithelial cells. (C,C') On sections consecutive to B and B', expression of E-cad protein is detected in heterozygous teratomas (C), and is absent in homozygous Ncad-GFP teratomas (C'). (D,D') Epithelia are formed in heterozygous (D) and homozygous Ncad-GFP teratomas (D') and are positive for cytokeratin 8 immunolabeling. Scale bar: 100 µm.