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. 2014 Aug 25;30(4):410-22.
doi: 10.1016/j.devcel.2014.06.019. Epub 2014 Aug 7.

Notch and hippo converge on Cdx2 to specify the trophectoderm lineage in the mouse blastocyst

Teresa Rayon  1 , Sergio Menchero  1 , Andres Nieto  2 , Panagiotis Xenopoulos  3 , Miguel Crespo  1 , Katie Cockburn  4 , Susana Cañon  1 , Hiroshi Sasaki  5 , Anna-Katerina Hadjantonakis  3 , Jose Luis de la Pompa  6 , Janet Rossant  4 , Miguel Manzanares  7
Affiliations

Notch and hippo converge on Cdx2 to specify the trophectoderm lineage in the mouse blastocyst

Teresa Rayon et al. Dev Cell. .

Abstract

The first lineage choice in mammalian embryogenesis is that between the trophectoderm, which gives rise to the trophoblast of the placenta, and the inner cell mass, from which is derived the embryo proper and the yolk sac. The establishment of these lineages is preceded by the inside-versus-outside positioning of cells in the early embryo and stochastic expression of key transcription factors, which is then resolved into lineage-restricted expression. The regulatory inputs that drive this restriction and how they relate to cell position are largely unknown. Here, we show an unsuspected role of Notch signaling in regulating trophectoderm-specific expression of Cdx2 in cooperation with TEAD4. Notch activity is restricted to outer cells and is able to influence positional allocation of blastomeres, mediating preferential localization to the trophectoderm. Our results show that multiple signaling inputs at preimplantation stages specify the first embryonic lineages.

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Figures

Figure 1
Figure 1. A cis-regulatory element upstream of Cdx2 drives restricted expression in the trophectoderm
(A–D) H2BmRFP reporter expression driven by the Cdx2 trophectoderm enhancer (TEE) at (A) the non-compacted 8-cell stage, (B) compacted 8-cell morula stage, (C) 16-cell stage, and (D) blastocyst stage. H2BmRFP was detected by immunohistochemistry and nuclei were stained with DAPI. Scale bars, 10 μm. (E) Quantification of the number of TEE-positive cells (y-axis) per embryo, staged by total cell number (x-axis). Each dot represents an individual embryo, and the bar and whiskers indicate means and standard deviations. (F) Correlation of TEE activity with endogenous CDX2 protein expression at the 8-cell (n=24, 3 embryos) and 16-cell stages (n=80, 5 embryos). Individual blastomeres were scored for H2BmRFP and CDX2 immunostaining. See also Figure S1.
Figure 2
Figure 2. The Hippo pathway is not sufficient to regulate the Cdx2 TEE
(A–B) TEE activity (red) and TEAD4 immunodetection (green) in (A) wild type (wt) and (B) Tead4 knockout embryos (Tead−/−). Nuclei were stained with DAPI (blue). Maximal projections (max. proj) of merged images are shown in the right panels. (C) Immunohistochemistry for CDX2 (green) in two Tead4 mutant embryos from the same litter. Nuclei were stained with DAPI (blue). Scale bars, 10 μm. (D) Relative expression of Cdx2, Oct4 and Nanog in the 5TVER7 cell line in response to tamoxifen (Tx) induction in EMFI-CM medium of TEAD4-mediated transdifferentiation of ES to TS cells. (E) Percentage of 5TVER7 cells that activate the TEE in response to tamoxifen (Tx) induction in EMFI-CM medium of TEAD4-mediated transdifferentiation of ES to TS cells. Data are means ± s.e.m. n=3. **p<0.01, *p<0.05 by Student’s t-test. (F) TEE activity (red) and GFP expression (green) in blastocysts injected with Lats2-GFP mRNA. White arrowheads mark TEE+ nuclei in which Lats2-GFP is expressed. Nuclei were stained with DAPI (blue). A merged image is shown in the right panel. See also Figure S2.
Figure 3
Figure 3. The Notch signaling pathway is active in the trophectoderm
(A–C) CBF:H2B-Venus reporter expression (green) at (A) the non-compacted 8-cell stage, (B) morula stage, and (C) blastocyst stage. (D) Treatment of embryos form the CBF:H2B-Venus line with the γ-secretase inhibitor RO4929097 (RO) downregulates reporter activity. (E) Immunodetection of Notch1 intracellular domain (N1ICD, red). Nuclei were stained with DAPI. Scale bars, 10 μm. See also Figure S3.
Figure 4
Figure 4. RBPJ and TEAD binding sites are necessary for the activity of the Cdx2 TEE
(A) Diagram of the Cdx2 locus showing the TEE fragment with the putative sites for RBPJ (blue) and for TEAD (green) and the corresponding mutated versions. (B) Luciferase activity of wild type and mutated versions of the TEE (TEERBPJmut, TEETEADmut and TEERBPJ/TEADmut) in response to overexpression of active N1ICD and/or TEAD4 (TeadVP16) in tissue culture assays. A construct carrying ten copies of a consensus RBPJ binding site (10xRBPJ) was used as a positive control. Data are means ± s.e.m. n=8 10xRBPJ), 7 (TEE), 5 (TEERBPJmut), 4 (TEETEADmut) and 4 (TEERBPJ/TEADmut). **p<0.01, *p<0.05 by Bonferroni post test; n.s., not significant. (C) Activity of wild type TEE, (D) TEERBPJmut, (E) TEETEADmut and (F) TEERBPJ/TEADmut in transient transgenic embryos. A representative transgenic embryo is shown for each construct (red, mRFP; blue, DAPI). Scale bars, 10 μm. (G) Diagram of the experimental setup of inhibitor versus DMSO treatment of embryos from the same batch of microinjected embryos. (H) Percentage of transgenic expression (TE, TE + ICM, or weak) in embryos microinjected with the wildtype (n=30 transgenic/150 total), TEETEADmut (n=91/359), TEERBPJmut (n=66/296) or TEERBPJ/TEADmut (n= 9/79) versions of the TEE and treated with the indicated inhibitor (RO, RO49229097; VP, Verteporfin). **p<0.01, ***p<0.001 by Chi squared test. See also Figure S4.
Figure 5
Figure 5. TEE activity and Cdx2 expression require transcriptional inputs from Notch and TEAD4
(A–E) TEE activity (red) and CDX2 immunodetection (green) in (A) Tead4+/−, (B) Rbpj+/−, (C) Rbpj−/−, (D) Rbpj+/−;Tead4+/−, and (E) Rbpj−/−;Tead4+/− mutant embryos. Nuclei were stained with DAPI. Arrowheads in C–E indicate TEE-;Cdx2+ outer blastomeres. Maximal projections of merged images are shown in the right panels. Scale bars, 10 μm. (F) Quantified CDX2 expression in outer cells of wild type blastocysts (n=89, 3 embryos) and in Tead4+/− (n=140, 3 embryos), Rbpj+/− (n=128, 4 embryos), Rbpj−/− (n=150, 4 embryos), Rbpj+/−;Tead4+/− (n=115, 4 embryos) and Rbpj−/−;Tead4+/− allelic combinations (n=159, 3 embryos). Boxes span the 25th to the 75th percentile, internal horizontal lines indicate median values, and whiskers show minima and maxima. ***p<0.001 by Bonferroni post test. (G) Relative expression of Cdx2, Gata3, Eomes, Oct4 and Nanog in pools of 25 embryos (n=6) treated from 2-cell until blastocyst stage with DMSO or RO. Data are means ± s.e.m. **p<0.01 by Student’s t-test. See also Figure S5.
Figure 6
Figure 6. Notch regulates Cdx2 expression and instructs cells to adopt an outer position in the blastocyst
(A) Expression of the reporter from the R26-stop-N1ICD-ires-EGFP line (green) and CDX2 (red) when recombined by maternal Sox2-Cre. White arrowheads mark non-recombined cells. Nuclei were stained with DAPI. Scale bars, 10 μm. (B) Quantified CDX2 expression in all N1ICD-overexpressing (N1OE+; green) and non-overexpressing (N1OE−) blastomeres in embryos generated from the ♂R26-stop-N1ICD-ires-EGFP X ♀Sox2-Cre cross (n=313 N1OE+; 93 N1OE− blastomeres/10 embryos). (C) Quantified CDX2 expression in outer N1OE+ (n=247) and N1OE− (n=59) blastomeres. In (B and C), boxes span the 25th to the 75th percentile, internal horizontal lines indicate median values, and whiskers show minima and maxima. ***p<0.001 by Student’s t-test. (D) Inside/outside distribution of all N1OE+ and N1OE− blastomeres compared with the distribution in wild type (wt) blastomeres (n=304, 6 embryos). ***p<0.001 by Chi squared test. (E) Distribution per embryo of inside (grey) and outside cells (white) in wild type (wt; n=6) and N1ICD over-expressing embryos (N1OE; n=10). For N1OE embryos, the contribution of EGFP-positive cells (green) to each population is also shown. See also Figure S6.
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