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First published online 11 August 2004
doi: 10.1242/dev.01316

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Maternal ß-catenin and E-cadherin in mouse development

¹ The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA
² Max-Planck Institute of Immunobiology, Stuebeweg 51, D-79108 Freiburg, Germany

View larger version (12K): [in a new window]   Fig. 1. Mating scheme used to generate embryos lacking either maternal E-cadherin or ß-catenin. To generate females whose oocytes would be deficient in either ß-catenin or E-cadherin, homozygous floxed females (Floxed/Floxed: [ßF/ßF] or [EF/EF]) of each line were crossed with homozygous C57BL/6-Tg(Zp3-cre)93Knw males (cre/cre) containing a cre-recombinase transgene under control of the Zp3 promoter. Males hemizygous for the Zp3-cre transgene and heterozygous for either floxed allele (Floxed/+;cre/Ø: [ßF/ß;cre/Ø] or [EF/E;cre/Ø]), were backcrossed to females homozygous for the ß-catenin or E-cadherin floxed alleles (Floxed/Floxed: [ßF/ßF] or [EF/EF]). This generated females homozygous for either floxed allele and heterozygous for Zp3-cre (Floxed/Floxed;cre/Ø: [ßF/ßF;cre/Ø] or [EF/EF;cre/Ø]) that produced oocytes deficient in either ß-catenin or E-cadherin. These females were crossed to wild-type C57BL/6J males to generate embryos lacking maternal ß-catenin or E-cadherin, but with a wild-type paternal allele (Floxeddel/+; del=deleted floxed). Control females homozygous for either floxed allele, but not carrying the Zp3-cre transgene (Floxed/+: [ßF/ßF;Ø/Ø] or [EF/EF;Ø/Ø]) were mated to wild-type C57BL/6J males (+/+; Ø;Ø) to generate control embryos. To determine whether presence of the Zp3-cre transgene would influence the outcome of embryo development, control females heterozygous for either floxed allele and hemizygous for Zp3-cre (Floxed/+;cre/Ø: [ßF/ß;cre/Ø] or [EF/E;cre/Ø]) were also mated to wild-type C57BL/6J males. Mice homozygous for both E-cadherin and ß-catenin floxed alleles were bred by intercrossing mice homozygous for either ß-catenin or E-cadherin floxed alleles ([ßF/ßF] or [EF/EF]), and crossing their offspring inter se. To obtain females homozygous for both floxed alleles and hemizygous for the Zp3-cre transgene, females homozygous for both floxed alleles were crossed to males homozygous for both floxed alleles, and hemizygous for the Zp3-cre transgene [ßF/ßF;EF/EF;cre/Ø]. This cross produced [ßF/ßF;EF/EF;cre/Ø] females that gave rise to oocytes deficient in both ß-catenin and E-cadherin.

View larger version (26K): [in a new window]   Fig. 2. Effective elimination of E-cadherin and ß-catenin sequences encompassed by loxP sites in oocytes, and characteristics of the floxed allele of ß-catenin. (A) Genotypes of some pups from females producing oocytes lacking E-cadherin. The PCR product characterizing the floxed-deleted E-cadherin allele inherited from the mother is indicated. The internal control is an unrelated wild-type product used as an indicator of successful PCR reactions. Genotypes of the control DNA are indicated at the top of each lane. (B) Genotypes of some pups from females producing oocytes lacking ß-catenin. The upper, floxed-deleted ß-catenin allele inherited from the female is indicated, as well as the lower wild-type ß-catenin allele inherited from the male. The middle product representing the floxed allele is clearly absent in the DNA of the pups analyzed. Genotypes of control DNA are indicated at top of each lane. (C) RT-PCR using primers specific for the 3' sequences of ß-catenin encoding the C-terminal part of the protein. A PCR replicon is detectable in all the embryos ostensibly lacking maternal ß-catenin (i.e. O, Z and e2; ßF/ßF; cre/Ø; top panel), as well as in the control embryos (ßF/ßF; Ø/Ø; bottom panel). Mitochondrial ATP synthase (mt-Atp6) primers were used as a control in both cases. O, ovulated oocyte; Z, zygote; e2, early two-cell embryo; l2, late two-cell embryo; l4, late four-cell embryo; 5/7, five- to seven-cell embryo; 8, eight-cell embryo; M, morula; B, blastocyst. (D) Western blot analysis using the polyclonal ß-catenin antibody recognizing the C-terminal part of the protein. ßF/+ indicates extract obtained from a control animal containing one floxed ß-catenin allele and one wild-type allele. ßF-del/+ indicates extract from a heterozygous animal containing a deleted-floxed allele and a wild-type allele. ß-catenin, wild-type ß-catenin; –N-ß-catenin, 52 kDa protein recognized by the polyclonal ß-catenin antibody. (E) Schematic representation of the interaction of ß-catenin with different binding partners (not to scale). The N-terminal and C-terminal are depicted by rectangular black boxes with an N or C, respectively. The 12 armadillo repeats are depicted by square, numbered black boxes. The regions where specific proteins interact with ß-catenin are depicted by brackets, with the name of the protein indicated. The large gray box indicates the part of ß-catenin absent in the floxed ß-catenin (–N-ß-catenin or truncated) allele. Ebi, Ebi; ß-Trcp, ß-transducin repeat containing protein; Gsk3ß, glycogen synthase kinase 3ß; CKI, casein kinase I; APC, adenomatous polyposis coli; BRG1, SMARCA4; XSox3/17, Xenopus Sox3 or 17; ICAT, CATNBIP1, catenin beta interacting protein 1; CBP/p300, CREBBP, CREB binding protein/E1A binding protein p300; SDCCAG33, serologically defined colon cancer antigen 33.

View larger version (40K): [in a new window]   Fig. 3. (A) No ß-catenin is found in embryos expressing truncated ß-catenin, using an N-terminal specific antibody, until the paternal allele is activated. A monoclonal ß-catenin antibody that recognizes an epitope in the N-terminal part of the protein was used. Cre/Ø indicates embryos expressing truncated ß-catenin; +/+ indicates control embryos. ß-catenin synthesized from the paternal allele is first detected on the surface of embryos expressing truncated ß-catenin at the 4- to 8-cell stage transition (6/8-cell stage). (B) E-cadherin is absent in embryos prior to activation of the paternal allele. A polyclonal antibody against E-cadherin was used. E-cadherin is first detected on the surface of embryos lacking maternal E-cadherin at the late morula stage. Cre/Ø indicates embryos lacking maternal E-cadherin; +/+ indicates control embryos. The stage of embryonic development is indicated below the figure; fluorescent (FL) or Nomarski Differential Interference Contrast (DIC) images are indicated on the left. Scale bar: 10 µm.

View larger version (66K): [in a new window]   Fig. 4. Localization of E-cadherin and ß-catenin in embryos lacking the binding partner. (A) E-cadherin is detectable on the blastomere surface of embryos expressing truncated ß-catenin. E-cadherin is visible on the surface of the blastomeres of both the control embryos (+/+) and the embryos expressing truncated ß-catenin (Cre/Ø). E-cadherin is also detectable in the cytoplasm of 2-cell and 4-cell stage embryos expressing truncated ß-catenin. (B) ß-catenin is present in the nucleus of early embryos lacking maternal E-cadherin. ß-catenin was detected in control embryos (+/+) and embryos lacking maternal E-cadherin (Cre/Ø), using the polyclonal ß-catenin antibody recognizing the C terminus of the protein. ß-catenin is visible on the blastomere surface of control zygotes, 2-cell stage embryos and 8-cell stage embryos, with some ß-catenin also visible in the cytoplasm of control zygotes and 2-cell stage embryos. By contrast, ß-catenin is visible in the pronuclei and cytoplasm of zygotes, as well as in the nuclei and cytoplasm of 2-cell stage embryos lacking maternal E-cadherin. Only a small amount of ß-catenin is detectable in 8-cell embryos lacking maternal E-cadherin. (C) Truncated ß-catenin is present in the nucleus of embryos lacking maternal E-cadherin. ß-catenin was detected in control embryos (+/+), and in embryos expressing truncated ß-catenin and lacking maternal E-cadherin (double mutant) embryos (Cre/Ø), using the polyclonal ß-catenin antibody that detects the C-terminal part of the protein. ß-catenin is detected in the pronuclei of control and double-mutant zygotes, on the surface and in the cytoplasm of control 2-cell stage embryos, in the nuclei and cytoplasm of double-mutant 2-cell stage embryos, and on the surface of 4 to 8-cell stage control and double-mutant embryos. Note for B and C, blastomeres of 8-cell stage embryos lacking maternal E-cadherin do not adhere to each other. Consequently all manipulations during the immunostaining process were carried out with extreme caution to maintain blastomeres of embryos in a clump. No fluorescence was detected in embryos where the primary antibody was omitted (data not shown). Scale bars: 10 µm.

View larger version (28K): [in a new window]   Fig. 5. The level of ß-catenin in 2-cell stage embryos is controlled by the same mechanism as in somatic cells. (A) Involvement of the proteasome. The presence (+) or absence (–) of maternal E-cadherin (E-cadherinmat) in the embryos analyzed is indicated, as is the presence (+) or absence (–) of the proteasome inhibitor MG132 (5 µM). The monoclonal ß-catenin antibody (ß-catenin Ab) was used for staining (+), except where it was omitted (–) to determine background levels of staining. Secondary antibody was used in every staining. Scale bar: 10 µm. (B) Transcripts for proteins interacting with ß-catenin in the cytoplasm or nucleus are present in oocytes and early embryos. Names of the transcripts are indicated to the left of the figure.

View larger version (42K): [in a new window]   Fig. 6. (A) Embryos expressing truncated ß-catenin do not develop optimally, as indicated by fewer pups per litter. Bar graph depicting the mean number of pups per litter obtained from females producing oocytes expressing truncated ß-catenin (ßF/ßF;cre/Ø) and control females (ßF/ßF and ßF/ß;cre/Ø). The number of litters recorded (n) in each group is indicated at the bottom of the graph. (B,C) The paternal alleles of ß-catenin and E-cadherin are activated sequentially. (B) Expression of E-cadherin in embryos lacking maternal E-cadherin (top) and control embryos (bottom). (C) Expression of wild-type ß-catenin in embryos expressing truncated ß-catenin (top) and control embryos (bottom). The expression of both genes was monitored by RT-PCR and subsequent Southern blot analysis of the PCR products using a 32P-labeled E-cadherin cDNA probe. Wild-type ß-catenin was monitored using primers situated in sequences coding for the N-terminal part of the protein. The gene product monitored is indicated on top of the figure; the genotype of the females from which the embryos were isolated is indicated on the right. Mitochondrial ATP synthase (mt-Atp6) was used as a control for the RT-PCR, and was detected by ethidium bromide staining. Embryo stages are the same as in Fig. 2.

View larger version (35K): [in a new window]   Fig. 7. Schematic representation of the phenotypes obtained for embryos either expressing truncated ß-catenin, lacking maternal E-cadherin, or expressing truncated ß-catenin as well as lacking maternal E-cadherin. (A) A control early/late 2-cell stage embryo (E/L2C) where interaction of ß-catenin (yellow and blue rectangles) and E-cadherin (blue zig-zag lines) ensures adhesion of the two blastomeres. (B) In early 2-cell stage embryos (E2C) lacking maternal E-cadherin, the adhesion complex cannot form, resulting in blastomeres that are not able to adhere. Consequently larger amounts of ß-catenin translocate to the nucleus. (C) In early 2-cell embryos expressing truncated ß-catenin (yellow squares), the blastomeres fail to adhere, even though E-cadherin is present on the blastomere surface. (D) In late 2-cell embryos expressing truncated ß-catenin, the wild-type paternal ß-catenin allele is activated. Protein translated from this allele is sequestered by E-cadherin to form the adhesion complex (yellow and blue rectangles with blue zig-zag lines attached), and a lesser amount is translocated to the nucleus. (E) By contrast, in late 2-cell stage embryos expressing truncated ß-catenin and also lacking maternal E-cadherin, all newly synthesized ß-catenin is able to translocate to the nucleus because no E-cadherin is present to sequester it to the adhesion complex.