Defective trophoblast function in mice with a targeted mutation of Ets2

Abstract

Members of the Ets family of transcription factors mediate transcriptional responses of multiple signaling pathways in diverse cell types and organisms. Targeted deletion of the conserved DNA binding domain of the Ets2 transcription factor results in the retardation and death of homozygous mouse embryos before 8.5 days of embryonic development. Defects in extraembryonic tissue gene expression and function include deficient expression of matrix metalloproteinase-9 (MMP-9, gelatinase B), persistent extracellular matrix, and failure of ectoplacental cone proliferation. Mutant embryos were rescued by aggregation with tetraploid mouse embryos, which complement the developmental defects by providing functional extraembryonic tissues. Rescued Ets2-deficient mice are viable and fertile but have wavy hair, curly whiskers, and abnormal hair follicle shape and arrangement, resembling mice with mutations of the EGF receptor or its ligands. However, these mice are not deficient in the production of TGFalpha or the EGF receptor. Homozygous mutant cell lines respond mitogenically to TGFalpha, EGF, FGF1, and FGF2. However, FGF fails to induce MMP-13 (collagenase-3) and MMP-3 (stromelysin-1) in the Ets2-deficient fibroblasts. Ectopic expression of Ets2 in the deficient fibroblasts restores expression of both matrix metalloproteinases. Therefore, Ets2 is essential for placental function, mediating growth factor signaling to key target genes including MMP-3, MMP-9, and MMP-13 in different cell types, and for regulating hair development.

Figure 1

RNA and protein expression from the ets2^db1 targeted allele. (A) RNase protection analysis of RNA derived from ets2^db1/+ and ets2^db1/db1 fibroblast lines utilizing a DNA binding domain probe. (Lane 1) size markers (M); (lane 2) probes (P) for Ets2 (upper) and the L32 ribosomal protein RNA (lower); (lane 3) ets2^db1/+ (H) RNA; (lane 4) ets2^db1/db1 homozygous (db) RNA. (B) Northern blot analysis of 5 μg of poly(A)⁺ RNA from the same cell lines. A probe from the 5′ end of the ets2 cDNA detected ets2-related RNA in wild-type (W), heterozygous (H), and homozygous (db) cells. A neo probe detects RNA only in heterozygous ets2^db1/+ (lane 5) and ets2^db1/db1 RNA (lane 6). (C) Immunoprecipitation analysis of Ets2 and Ets2^db1 proteins. Ets2 antiserum (McCarthy et al. 1997) detects the wild-type Ets2 protein (lane 9, Ets2), the product of the ets2^db1 allele (lanes 7,10,14, Ets/neo), an epitope-tagged form of Ets2 (lane 10, FNets2) and an epitope-tagged form of the transactivation domain of Ets2 (lane 11, FNtad). No specific signal was detected with preimmune serum (lanes 1–3) or Ets2 antiserum incubated with excess recombinant Ets2 (lanes 4–6). Translation products of ets2 mRNA (lane 12) and the ets2/neo fusion transcript (lane 13). The supernant fraction (cyt) and solubilized pellet (nuc) fractions of ets2^db1/db1 cells were immunoprecipitated with Ets2 antiserum (lanes 14,15). The ets2^db1/db1, ets2^db1/+, and ets2^+/+ cell types are EKO1, EHT1, and 3T3 cells, respectively. E8 is a rescued clone of EKO1 that expressed the FNets2 protein (lane 10). (Lane 11) Control cells expressing FNtad. (D) Schematic representation of ets2 and ets2^db1 genes, RNAs, and proteins. The structures of the 3′ end of the wild-type and targeted genes are shown in the middle with solid boxes representing exons. The regions coding for the DNA-binding domain are shaded. The position of the pMC1neoA gene is labeled Neo with promoter/enhancer region represented as crosshatched. The predicted RNAs for the two genes are shown at the top and bottom. The expected 3′ noncoding region of both mRNAs are represented by the smaller open rectangles at the right. The proteins coded for by the RNAs are indicted by the arrows with the expected size indicated.

Figure 1

RNA and protein expression from the ets2^db1 targeted allele. (A) RNase protection analysis of RNA derived from ets2^db1/+ and ets2^db1/db1 fibroblast lines utilizing a DNA binding domain probe. (Lane 1) size markers (M); (lane 2) probes (P) for Ets2 (upper) and the L32 ribosomal protein RNA (lower); (lane 3) ets2^db1/+ (H) RNA; (lane 4) ets2^db1/db1 homozygous (db) RNA. (B) Northern blot analysis of 5 μg of poly(A)⁺ RNA from the same cell lines. A probe from the 5′ end of the ets2 cDNA detected ets2-related RNA in wild-type (W), heterozygous (H), and homozygous (db) cells. A neo probe detects RNA only in heterozygous ets2^db1/+ (lane 5) and ets2^db1/db1 RNA (lane 6). (C) Immunoprecipitation analysis of Ets2 and Ets2^db1 proteins. Ets2 antiserum (McCarthy et al. 1997) detects the wild-type Ets2 protein (lane 9, Ets2), the product of the ets2^db1 allele (lanes 7,10,14, Ets/neo), an epitope-tagged form of Ets2 (lane 10, FNets2) and an epitope-tagged form of the transactivation domain of Ets2 (lane 11, FNtad). No specific signal was detected with preimmune serum (lanes 1–3) or Ets2 antiserum incubated with excess recombinant Ets2 (lanes 4–6). Translation products of ets2 mRNA (lane 12) and the ets2/neo fusion transcript (lane 13). The supernant fraction (cyt) and solubilized pellet (nuc) fractions of ets2^db1/db1 cells were immunoprecipitated with Ets2 antiserum (lanes 14,15). The ets2^db1/db1, ets2^db1/+, and ets2^+/+ cell types are EKO1, EHT1, and 3T3 cells, respectively. E8 is a rescued clone of EKO1 that expressed the FNets2 protein (lane 10). (Lane 11) Control cells expressing FNtad. (D) Schematic representation of ets2 and ets2^db1 genes, RNAs, and proteins. The structures of the 3′ end of the wild-type and targeted genes are shown in the middle with solid boxes representing exons. The regions coding for the DNA-binding domain are shaded. The position of the pMC1neoA gene is labeled Neo with promoter/enhancer region represented as crosshatched. The predicted RNAs for the two genes are shown at the top and bottom. The expected 3′ noncoding region of both mRNAs are represented by the smaller open rectangles at the right. The proteins coded for by the RNAs are indicted by the arrows with the expected size indicated.

Figure 1

RNA and protein expression from the ets2^db1 targeted allele. (A) RNase protection analysis of RNA derived from ets2^db1/+ and ets2^db1/db1 fibroblast lines utilizing a DNA binding domain probe. (Lane 1) size markers (M); (lane 2) probes (P) for Ets2 (upper) and the L32 ribosomal protein RNA (lower); (lane 3) ets2^db1/+ (H) RNA; (lane 4) ets2^db1/db1 homozygous (db) RNA. (B) Northern blot analysis of 5 μg of poly(A)⁺ RNA from the same cell lines. A probe from the 5′ end of the ets2 cDNA detected ets2-related RNA in wild-type (W), heterozygous (H), and homozygous (db) cells. A neo probe detects RNA only in heterozygous ets2^db1/+ (lane 5) and ets2^db1/db1 RNA (lane 6). (C) Immunoprecipitation analysis of Ets2 and Ets2^db1 proteins. Ets2 antiserum (McCarthy et al. 1997) detects the wild-type Ets2 protein (lane 9, Ets2), the product of the ets2^db1 allele (lanes 7,10,14, Ets/neo), an epitope-tagged form of Ets2 (lane 10, FNets2) and an epitope-tagged form of the transactivation domain of Ets2 (lane 11, FNtad). No specific signal was detected with preimmune serum (lanes 1–3) or Ets2 antiserum incubated with excess recombinant Ets2 (lanes 4–6). Translation products of ets2 mRNA (lane 12) and the ets2/neo fusion transcript (lane 13). The supernant fraction (cyt) and solubilized pellet (nuc) fractions of ets2^db1/db1 cells were immunoprecipitated with Ets2 antiserum (lanes 14,15). The ets2^db1/db1, ets2^db1/+, and ets2^+/+ cell types are EKO1, EHT1, and 3T3 cells, respectively. E8 is a rescued clone of EKO1 that expressed the FNets2 protein (lane 10). (Lane 11) Control cells expressing FNtad. (D) Schematic representation of ets2 and ets2^db1 genes, RNAs, and proteins. The structures of the 3′ end of the wild-type and targeted genes are shown in the middle with solid boxes representing exons. The regions coding for the DNA-binding domain are shaded. The position of the pMC1neoA gene is labeled Neo with promoter/enhancer region represented as crosshatched. The predicted RNAs for the two genes are shown at the top and bottom. The expected 3′ noncoding region of both mRNAs are represented by the smaller open rectangles at the right. The proteins coded for by the RNAs are indicted by the arrows with the expected size indicated.

Figure 1

RNA and protein expression from the ets2^db1 targeted allele. (A) RNase protection analysis of RNA derived from ets2^db1/+ and ets2^db1/db1 fibroblast lines utilizing a DNA binding domain probe. (Lane 1) size markers (M); (lane 2) probes (P) for Ets2 (upper) and the L32 ribosomal protein RNA (lower); (lane 3) ets2^db1/+ (H) RNA; (lane 4) ets2^db1/db1 homozygous (db) RNA. (B) Northern blot analysis of 5 μg of poly(A)⁺ RNA from the same cell lines. A probe from the 5′ end of the ets2 cDNA detected ets2-related RNA in wild-type (W), heterozygous (H), and homozygous (db) cells. A neo probe detects RNA only in heterozygous ets2^db1/+ (lane 5) and ets2^db1/db1 RNA (lane 6). (C) Immunoprecipitation analysis of Ets2 and Ets2^db1 proteins. Ets2 antiserum (McCarthy et al. 1997) detects the wild-type Ets2 protein (lane 9, Ets2), the product of the ets2^db1 allele (lanes 7,10,14, Ets/neo), an epitope-tagged form of Ets2 (lane 10, FNets2) and an epitope-tagged form of the transactivation domain of Ets2 (lane 11, FNtad). No specific signal was detected with preimmune serum (lanes 1–3) or Ets2 antiserum incubated with excess recombinant Ets2 (lanes 4–6). Translation products of ets2 mRNA (lane 12) and the ets2/neo fusion transcript (lane 13). The supernant fraction (cyt) and solubilized pellet (nuc) fractions of ets2^db1/db1 cells were immunoprecipitated with Ets2 antiserum (lanes 14,15). The ets2^db1/db1, ets2^db1/+, and ets2^+/+ cell types are EKO1, EHT1, and 3T3 cells, respectively. E8 is a rescued clone of EKO1 that expressed the FNets2 protein (lane 10). (Lane 11) Control cells expressing FNtad. (D) Schematic representation of ets2 and ets2^db1 genes, RNAs, and proteins. The structures of the 3′ end of the wild-type and targeted genes are shown in the middle with solid boxes representing exons. The regions coding for the DNA-binding domain are shaded. The position of the pMC1neoA gene is labeled Neo with promoter/enhancer region represented as crosshatched. The predicted RNAs for the two genes are shown at the top and bottom. The expected 3′ noncoding region of both mRNAs are represented by the smaller open rectangles at the right. The proteins coded for by the RNAs are indicted by the arrows with the expected size indicated.

Figure 2

In situ localization of ets2 RNA and retarded growth of ets2^db1/db1 embryos. Whole mount in situ analysis of ets2 expression in E6–E7.5 embryos. (A) At E6, prior to primitive streak (ps) formation, expression is seen in the extraembryonic ectoderm (exec) and trophectoderm (troph), but not in the overlying extraembryonic endoderm (exen). (B) Primitive streak stage at E6.5 and elongated at E7.5 (C). (D) Brachyury (T) expression at E7.5, used as a control. (E) Embryos resulting from a mating of ets2^db1/+ parents were dissected at E7.5 days. The four small embryos were subsequently identified as ets2^db1/db1. The trophoblastic tissue was removed from the normal sized embryos. (F) A higher magnification showing the unusual cone-shaped yolk sac of the arrested ets2^db1/db1 embryos.

Figure 3

Abnormal implantation in Ets2 mutant embryos. (A,D), normal implantation in E6.5 and E7.5 wild-type embryos in which the embryonic trophoblast cells migrate out from the ectoplacental cone (epc) to form a hybrid vasculature in contact with maternal blood. (B) The trophoblast cells of Ets2 mutant embryos fail to migrate and differentiate, resulting in the characteristic bloody implantation site. (C) A higher magnification of the E6.5 mutant embryo shows a membrane (arrow) covering the ectoplacental cone. (E) At E7.5, the mutant embryo is small and beginning to die. (F) High magnification of the E7.5 mutant shows apoptotic nuclei in embryonic ectoderm (arrow). Scale bars, 125 μm. Hematoxylin and eosin-stained glycol methacrylate sections.

Figure 4

Abnormal protein expression in ets2^db1/db1 embryos at E7.5. (A,B) Tissue-specific regulation of MMP-9 in a mutant embryo. Note the absence of immunoreactive MMP-9 in the surrounding trophoblast cells of the mutant (B), as compared to wild-type (A), but the area where primitive streak should form shows a positive stain (arrow) with anti-MMP-9. (C,D) Placental lactogen-1 staining is normal in wild-type (C) (brown color), but is substantially upregulated in the mutant embryo (D). (E,F) Anti-laminin staining for basement membranes (blue color) shows extended expression of laminin around the ectoplacental cone. (G,H) Anti-PECAM antibody to detect endothelial cells of blood vessels (blue color) shows that vascular connections to maternal circulation are severely compromised in the mutant embryo (H), compared to the wild type (G). The higher magnification inset in G shows the staining of ectoplacental cone cells. Primary antibodies were detected with HRP-labeled secondary antibodies, and then histochemical detection for peroxidase. A, B, E, F, G, and H are True Blue substrate with eosin counterstain. C and D are DAB (brown) substrate with methyl green counterstain.

Figure 5

Rescue of ets2^db1/db1 embryos by aggregation with tetraploid embryos. (A) E14.5-day embryos resulting from the aggregation with wild-type tetraploid embryos. (B) PCR analysis of the embryos reveals that embryo 1 is homozygous mutant, whereas embryos 3 and 4 are heterozygous and 2, 5, and 6 are wild-type. (C) PCR analysis of the yolk sac DNA. The equal intensity of the amplified bands of sample 1 indicates approximately equal contribution of wild-type and targeted cells to the yolk sac. (D,F) Three-week-old wild-type mice resulting from a tetraploid embryo aggregation experiment. (E,G) Rescued ets2^db1/db1 mouse. Note the wavy hair coat and rounded forehead (arrows).

Figure 6

Phenotype in whiskers, hair, and eye alteration of the ets2^db1/db1 adult mouse. (A,C,E,G,I) Wild-type mouse whiskers, eye, zigzag hair, whole mount skin, and hair follicle histology respectively; (B,D,F,H,J) from ets2^db1/db1 mouse. (D) Eye secretions of the ets2^db1/db1 mouse were transient and resolved spontaneously without treatment within a few weeks. (H) Arrows point to ingrown hairs lying wholly within the skin. View is from the lower part of the cleared skin in G and H. (J) Arrow points to abnormally oriented hair follicle. Note the unusual juxtaposition directly against the lower muscle layer of the skin.

Figure 7

RNase protection analysis of adult organ RNAs. (A) All RNAs were from male 90-day-old tetraploid embryo aggregation littermates except for brain and mammary gland samples of heterozygous and homozygous 50-day littermate females of a second tetraploid embryo aggregation experiment. The wild-type female mammary gland and brain samples were from a 90-day-old adult. Twenty or 10 μg (c-Jun, EGFr, Endo A, and Endo B) of total RNA from the indicated organs was analyzed by RNase protection with probes indicated at right. Protected fragments were resolved by acrylamide gel electrophoresis and detected by autoradiography in the presence of enhancer screens and quantified using a PhosphorImager. Images are from autoradiographic exposures of five different gels, each of which also contained L32 signals (additional data shown). Signals for uPA (lanes 4–9), HNF4, HNF3β (lanes 4–6) and EGFr, endo A, and endo B (lanes 16–18) are 1-day exposures for improved clarity. (B) Quantitation of ets1, MMP-3, MMP-9, uPA, and junB mRNA after normalization to L32 RNA in skin and mammary gland samples.

Figure 8

Role of Ets2 in FGF2 induction of MMP-13 and MMP-3 in cultured cells. The EKO1 Ets2-deficient fibroblastic cell line and two independent isolates that expressed transfected Ets2 (E8 and E13) were incubated in medium containing 0.5% fetal bovine serum for 2 days. FGF2 (50 ng/ml) was added for 7 hr and RNA was isolated. The indicated RNAs were detected by RNase protection analysis. Collagen IV is indicated by Col4. Shown are autoradiographic exposures of the acrylamide gels used to separate the protected fragments. The time of growth factor exposure is indicated in hours at the top of each lane. Note the increased expression of the MMP-3 (A, lanes 3,5) ) and MMP-13 (B, lanes 3,5) RNAs in the E8 and E13 cell lines and their further increase after 7 hr.