Initiation of a conserved trophectoderm program in human, cow and mouse embryos

Abstract

Current understandings of cell specification in early mammalian pre-implantation development are based mainly on mouse studies. The first lineage differentiation event occurs at the morula stage, with outer cells initiating a trophectoderm (TE) placental progenitor program. The inner cell mass arises from inner cells during subsequent developmental stages and comprises precursor cells of the embryo proper and yolk sac¹. Recent gene-expression analyses suggest that the mechanisms that regulate early lineage specification in the mouse may differ in other mammals, including human^2-5 and cow⁶. Here we show the evolutionary conservation of a molecular cascade that initiates TE segregation in human, cow and mouse embryos. At the morula stage, outer cells acquire an apical-basal cell polarity, with expression of atypical protein kinase C (aPKC) at the contact-free domain, nuclear expression of Hippo signalling pathway effectors and restricted expression of TE-associated factors such as GATA3, which suggests initiation of a TE program. Furthermore, we demonstrate that inhibition of aPKC by small-molecule pharmacological modulation or Trim-Away protein depletion impairs TE initiation at the morula stage. Our comparative embryology analysis provides insights into early lineage specification and suggests that a similar mechanism initiates a TE program in human, cow and mouse embryos.

Extended Data Figure 1

a, Representative images of mouse, cow and human embryos at each developmental stage analyzed. Yellow arrows point to blastomere flattening. Yellow outline remarks the compacted morula. Yellow arrowheads mark microlumens. Yellow asterisks show single dominant cavity. Yellow bars show zona pellucida (ZP) thinning. Embryos are not shown to scale. dpf = days post fertilization. b, c, Morphokinetic analysis of mouse, cow and human preimplantation development showing relative time in hours (b) and in percentage (c) (from 8-cell stage to the end of cavitation). d, Morphokinetic analysis for each of the mouse, cow and human preimplantation embryos used in the analysis, showing relative time in percentage (from the 8-cell stage up to the end of cavitation). n = 15 embryos for mouse and cow, and n = 16 for human. e, Quantification of the number of individual cells that divided between 8-cell stage and start of compaction in mouse, cow and human embryos. n = 15 embryos for mouse and human, and n = 14 for cow. f, Quantification of the total cell number in mouse (32-cell stage), cow and human morula stage embryos. n = 25 for mouse, n = 20 for cow and n = 34 for human. g, Quantification of the number of inner and outer cells in percentage in mouse, cow and human morula. n = 25 for mouse, n = 20 for cow and n = 34 for human. h, Quantification of the number of ICM and TE cells in percentage in mouse, cow and human blastocysts. n = 14 for mouse, n = 10 for cow and n = 12 for human. Mann Whitney U test, *** P < 0.001, **** P < 0.0001, ns = not significant.

Extended Data Figure 2

a, b, Immunofluorescence analysis of F-ACTIN (red), TEAD4 (magenta) and HOECHST-33342 nuclear staining (blue) in human morula embryo. Yellow arrowheads point to a cell without TEAD4 expression. c, Percentage of TEAD4-positive and -negative cells in human morula embryos (n = 112 cells from 5 embryos). d, Quantification of TEAD4 fluorescence intensity, normalized to HOECHST-33342 intensity, in either inner or outer cells in human morula embryos (n = 63 cells from 5 embryos). t-test, ns = not significant. e, Immunofluorescence analysis of GATA3 (green), E-CADHERIN (red), YAP1 (magenta) and DAPI nuclear staining (blue) in mouse morula stage embryos. f, g, Quantification of YAP1 (f) and GATA3 (g) fluorescence intensity, normalized to DAPI intensity, in either inner or outer cells in mouse morula stage embryos (n = 182 cells for YAP1 from 13 embryos and n = 139 cells for GATA3 from 9 embryos). t-test, ****p < 0.0001. h, Immunofluorescence analysis using various secondary-antibodies used in this paper and DAPI nuclear staining (blue) in mouse morula stage embryos (n = 3). i, Time-course immunofluorescence analysis of GATA3 (green), β-CATENIN (red), YAP1 (magenta) and DAPI nuclear staining (blue) in cow embryos at different developmental stages: pre-compaction (n = 5) and late compaction (n = 5), morula (n = 11) and expanded blastocyst (n = 5). j,k, Quantification of YAP1 (j) and GATA3 (k) fluorescence intensity, normalized to DAPI intensity, in either inner or outer cells in cow morula stage embryos (n = 97 cells from 11 embryos). t-test, ****p < 0.0001. l, Immunofluorescence analysis of GATA3 (green), GATA2 (red) and DAPI nuclear staining (blue) in a human morula stage embryo (n = 3). m, Immunofluorescence analysis of GATA3 (green), OCT4 (red), GATA2 (magenta) and DAPI nuclear staining (blue) in a human blastocyst stage embryo (n = 3). Yellow arrowheads point to outer cells expressing GATA3, while cyan arrow points to an inner cell devoid of GATA3 expression. Scale bars, as displayed in figures.

Extended Data Figure 3

a, Immunofluorescence analysis of YAP1 (green), F-ACTIN (red), WWTR1 (magenta) and DAPI nuclear staining (blue) in a human morula stage embryo (n = 3). b, Immunofluorescence analysis of YAP1 (green), NANOG (red) and WWTR1 (magenta) and DAPI nuclear staining (blue) in a human blastocyst stage embryo (n = 3). Yellow arrowheads point to outer and TE cells co-expressing YAP1 and WWTR1. c, Immunofluorescence analysis of GATA3 (green), KRT18 (magenta) and HOECHST-33342 nuclear staining (blue) in a human blastocyst stage embryo (n = 3). d, Genome browser view of the ATAC-seq signal at the GRHL2 locus. High confidence peaks (FDR < 0.001) were used to identify transcription factor motifs. Representative binding motifs associated with the footprints are highlighted. The average expression of GRHL2 in high GATA3-high and GATA3-low expressing cells at the morula is shown and the TPM units indicated. Scale bars, as displayed in figures. e, Immunofluorescence analysis of GRHL2 (green), F-ACTIN (red), GATA3 (magenta) and DAPI nuclear staining (blue) in human morula stage embryos (n = 3). Yellow arrowheads point to outer cells co-expressing GRHL2 and GATA3, and cyan arrows point to inner cells expressing GRHL2. f, Immunofluorescence analysis of GRHL2 (green), OCT4 (red), GATA3 (magenta) and DAPI nuclear staining (blue) in human expanded and hatching blastocyst stage embryo (n = 3 each stage). Scale bars, as displayed in figures.

Extended Data Figure 4

a-d, Scatter plots showing positive correlation of GATA3 expression profile with GRHL2, CLDN4, RAB20 (a), PTGES, TFEB, PLAC8 (b), ATP6V1B1, FXYD4, SLC7A2 (c), and VGLL4 (d) expression profiles in human morula cells. n = 197 cells. r = Pearson correlation coefficient. Values are displayed as log-transformed size-factor-normalized counts. The black line corresponds to a linear regression model fitted to the data with 95% confidence bands. e, Scatter plots of selected genes implicated in embryonic stem cell pluripotency and/or enriched in EPI/ICM precursors in human morula cells that were identified as negatively correlated with GATA3 expression. n = 197 cells. r = Pearson correlation coefficient. Values are displayed as log-transformed size-factor-normalized counts. The black line corresponds to a linear regression model fitted to the data with 95% confidence bands. f, Boxplots representing genes coincidently highly expressed in GATA3-high versus GATA3-low cells in human morula cells. Data are shown as Transcripts Per Million (TPM) + 1. Boxes correspond to the first and third quartiles, horizontal lines to the median, whiskers extend to 1.5 times the interquartile range and dots are outliers.

Extended Data Figure 5

a, Immunofluorescence analysis of SOX2 (green), E-CADHERIN (red), GATA3 (magenta) and DAPI nuclear staining (blue) in mouse morula stage embryos. b, Quantification of SOX2 fluorescence intensity, normalized to DAPI intensity, in either inner or outer cells in mouse morula stage embryos (n = 93 cells from 7 embryos). Yellow arrows mark outer cells expressing only GATA3. c, Time-course immunofluorescence analysis of SOX2 (green), β-CATENIN (red), GATA3 (magenta) and DAPI nuclear staining (blue) in cow embryos at pre-compaction (n = 5), late compaction (n = 5), morula (n = 9) and expanded blastocyst (n = 5) stages. d, Immunofluorescence analysis of SOX2 (green), β-CATENIN (red), GATA3 (magenta) and DAPI nuclear staining (blue) in human pre-compaction (n = 5) and late-compaction (n = 5) stage embryos. e, Quantification of SOX2 fluorescence intensity, normalized to DAPI intensity, in either inner or outer cells in cow morula stage embryos (n = 136 cells from 9 embryos). f, Quantification of SOX2 fluorescence intensity, normalized to DAPI intensity, in either inner or outer cells in human morula stage embryos (n = 68 cells from 6 embryos). Yellow arrowheads point to outer cells expressing SOX2 and GATA3. t-test, ****p < 0.0001, ns = not significant. g, Immunofluorescence analysis of SOX2 (green), F-ACTIN (red), GATA3 (magenta) and DAPI nuclear staining (blue) in human morula and blastocyst stage embryo (n = 3 each stage). The SOX2 antibody used in panel g in this figure is MAB2018 (R&D), while the one used in Figure 2 is 14-9811-82 (Ebioscience), both show a consistent signal for SOX2. Yellow arrowheads point to outer cells co-expressing GATA3 and SOX2, while cyan arrow points to an inner cell showing SOX2 expression only. Scale bar, as displayed in figure.

Extended Data Figure 6

a, Immunofluorescence analysis of aPKC (green), E-CADHERIN (red), AMOT (magenta) and DAPI nuclear staining (blue) in mouse morula stage embryos (n = 10). b, Fluorescence intensity profile of aPKC and AMOT shown along the yellow arrows in mouse morula stage embryos. c, Immunofluorescence analysis of aPKC (green), E-CADHERIN (red) and DAPI nuclear staining (blue) in mouse morula stage embryo. d, Immunofluorescence analysis of aPKC (green), β-CATENIN (red), AMOT (magenta) and DAPI nuclear staining (blue) in cow morula stage embryos (n = 10). e, Fluorescence intensity profile of aPKC and AMOT shown along the yellow arrows in cow morula stage embryos. f, Immunofluorescence analysis of aPKC (green) and DAPI nuclear staining (blue) in cow morula stage embryo (n = 3). g, Immunofluorescence analysis of aPKC (green), E-CADHERIN (red) and DAPI nuclear staining (blue) in human morula stage embryo (n = 3). aPKC antibody used in this figure panel c, f, g is LC-C354069 (LSBio), while aPKC antibody used in Figure 3 and in this figure panel a, d is sc-17781 (Santa Cruz). Both antibodies show strong aPKC apical expression. h, Immunofluorescence analysis of PARD6B (green), F-ACTIN (red), aPKC (magenta) and DAPI nuclear staining (blue) in human morula stage embryo (n = 3). i, Immunofluorescence analysis of PARD6B (green), NANOG (red), aPKC (magenta) and DAPI nuclear staining (blue) in human blastocyst stage embryo (n = 3). Scale bar, as displayed in figure.

Extended Data Figure 7

a, Schematic of aPKC inhibitor treatment in mouse embryos. b, Immunofluorescence analysis of GATA3 (green), E-CADHERIN (red), YAP1 (magenta) and DAPI nuclear staining (blue) in mouse morula stage embryos treated with different concentration of aPKC inhibitor (n = reported in Supplementary Table 6 for each condition). c, Schematic of aPKC inhibitor treatment in cow embryos. d, Immunofluorescence analysis of β-CATENIN (red), YAP1 (magenta) and DAPI nuclear staining (blue) in cow morula stage embryos treated with different concentration of aPKC inhibitor (n = reported in Supplementary Table 7 for each condition). e, Schematic of aPKC inhibitor treatment in human embryos. f, Immunofluorescence analysis of GATA3 (green), β-CATENIN (red), YAP1 (magenta) and DAPI nuclear staining (blue) in human morula stage embryos treated with different concentration of aPKC inhibitor (n = reported in Supplementary Table 8 for each condition). Scale bars, as displayed in figures.

Extended Data Figure 8

a, Immunofluorescence analysis of SOX2 (green), E-CADHERIN (red), YAP1 (magenta) and DAPI nuclear staining (blue) in control and aPKC inhibitor-treated mouse morula stage embryos. Yellow arrowheads point to outer cells expressing SOX2 in a mouse morula stage embryo. b, c, Quantification of SOX2 (b) and YAP1 (c) fluorescence intensity, normalized to DAPI intensity, in outer cells in control and aPKC-inhibitor treated mouse morula stage embryos (n = 155 cells from 19 embryos). t-test, ***p < 0.001, ****p < 0.0001. d, Schematic of aPKC inhibitor treatment in mouse embryos. e, Morphokinetic analysis of control and aPKC-treated mouse embryos showing relative time in percentage (from 2-cell stage to morula stage) (n = 88). f, Schematic of aPKC inhibitor treatment in mouse embryos. g, Quantification of percentage of mouse embryos either developing to form an expanded blastocyst or arrested morula in control (n = 30) and aPKC-inhibitor treated (n = 30) embryos. h, Schematic of aPKC inhibitor treatment in cow embryos. i, Quantification of percentage of cow embryos either developing to form an expanded blastocyst or arrested morula in control (n = 10) and aPKC-inhibitor treated (n = 10) embryos. j, Schematic of aPKC inhibitor treatment in human embryos. k, Quantification of percentage of human embryos either developing to form an expanded blastocyst or arrested morula in control (n = 12) and aPKC-inhibitor treated (n = 12) embryos. l, Immunofluorescence analysis of SOX2 (green), β-CATENIN (red), YAP1 (magenta) and DAPI nuclear staining (blue) in control and aPKC inhibitor-treated cow morula stage embryos. m, n, Quantification of SOX2 (m) and YAP1 (n) fluorescence intensity, normalized to DAPI intensity, in outer cells in control and aPKC-inhibitor treated cow morula stage embryos (n = 218 cells from 15 embryos). t-test, ****p < 0.0001, ns = not significant. o, Immunofluorescence analysis of SOX2 (green), F-ACTIN (red), YAP1 (magenta) and DAPI nuclear staining (blue) in control and aPKC inhibitor-treated human morula stage embryos. p, q, Quantification of SOX2 (p) and YAP1 (q) fluorescence intensity, normalized to DAPI intensity, in outer cells in control and aPKC-inhibitor treated cow morula stage embryos (n = 140 cells from 10 embryos). t-test, ****p < 0.0001, ns = not significant. Scale bars, as displayed in figures.

Extended Data Figure 9

a, Immunofluorescence analysis of TEAD4 (green), E-CADHERIN (red), YAP1 (magenta) and DAPI nuclear staining (blue) in control and aPKC inhibitor-treated mouse morula stage embryos. b, c, Quantification of TEAD4 (b) and YAP1 (c) fluorescence intensity, normalized to DAPI intensity, in outer cells in control and aPKC-inhibitor treated mouse morula stage embryos (n = 101 cells from 10 embryos). t-test, ****p < 0.0001, ns = not significant. d, Immunofluorescence analysis of TEAD4 (green), F-ACTIN (red), YAP1 (magenta) and DAPI nuclear staining (blue) in control and aPKC inhibitor-treated human morula stage embryos. e, f, Quantification of TEAD4 (e) and YAP1 (f) fluorescence intensity, normalized to DAPI intensity, in outer cells in control and aPKC-inhibitor treated human morula stage embryos (n = 89 cells from 6 embryos). t-test, ****p < 0.0001, ns = not significant. g, Schematic of the TRIM-Away approach. h, Schematic representation of the TRIM-Away experiment in mouse. i, Schematic representation of the two different antibodies used in the TRIM-Away experiment. j, Immunofluorescence analysis of anti-mouse secondary antibody to detect the electroporated aPKC antibody (green), YAP1 (red), GATA3 (magenta) and DAPI nuclear staining (blue) at the morula stage in control embryos and in embryos electroporated with mCherry-TRIM21 mRNA and anti-aPKC antibody (n = reported in Supplementary Table 9 for each condition). Scale bars, as displayed in figures.

Extended Data Figure 10

a, Immunofluorescence analysis of E-CADHERIN (green), mCHERRY (magenta) and DAPI nuclear staining (blue) at the morula stage in control embryos and in embryos electroporated with mCherry-TRIM21 mRNA and mouse anti-aPKC antibody (n = 10). b, Immunofluorescence analysis of anti-mouse secondary antibody to detect the electroporated aPKC antibody (green), E-CADHERIN (red), rabbit anti-aPKC to detect the aPKC protein (magenta) and DAPI nuclear staining (blue) in at the morula stage in embryos either electroporated with mCherry-TRIM21 mRNA only or with mCherry-TRIM21 mRNA and mouse anti-aPKC antibody. c, Quantification of aPKC cortical intensity (based on rabbit anti-aPKC signal) in outer cells at the morula stage in embryos electroporated either with mCherry-TRIM21 mRNA only or with mCherry-TRIM21 mRNA and mouse anti-aPKC antibody (n = 270 cells from 30 embryos). d, Immunofluorescence analysis of SOX2 (green), rabbit anti-aPKC to detect the aPKC protein (red), anti-mouse secondary antibody to detect the electroporated aPKC antibody (magenta) and DAPI nuclear staining (blue) at the morula stage in embryos electroporated either with mCherry-TRIM21 mRNA only or with mCherry-TRIM21 mRNA and mouse anti-aPKC antibody. Yellow arrowheads point to outer cells expressing SOX2 in a mouse morula stage embryo. 2 independent experiments. e, Quantification of SOX2 fluorescence intensity, normalized to DAPI intensity, in outer cells at the morula stage in embryos electroporated either with mCherry-TRIM21 mRNA only or with mCherry-TRIM21 mRNA and mouse anti-aPKC antibody (n = 120 cells 18 embryos). t-test, *p < 0.05. f, Immunofluorescence analysis of TEAD4 (green), anti-mouse secondary antibody to detect the electroporated aPKC antibody (magenta) and DAPI nuclear staining (blue) at the morula stage in embryos electroporated either with mCherry-TRIM21 mRNA only or with mCherry-TRIM21 mRNA and mouse anti-aPKC antibody. 2 independent experiments. g, Quantification of TEAD4 fluorescence intensity, normalized to DAPI intensity, in outer cells at the morula stage in embryos electroporated either with mCherry-TRIM21 mRNA only or with mCherry-TRIM21 mRNA and mouse anti-aPKC antibody (n = 90 cells from 10 embryos). t-test, ns = not significant. Scale bars as displayed in figures.

Extended Data Figure 11

a, Schematic representation of the TRIM-Away experiment. b, Immunofluorescence analysis of anti-mouse secondary antibody to detect the electroporated aPKC antibody (green), rabbit anti-aPKC to detect the aPKC protein (red), GATA3 (magenta) and DAPI nuclear staining (blue) in at the morula stage in human embryos either electroporated with mCherry-TRIM21 mRNA only or with mCherry-TRIM21 mRNA and mouse anti-aPKC antibody. c, Quantification of aPKC cortical intensity (based on rabbit anti-aPKC signal) in outer cells at the morula stage in human embryos electroporated either with mCherry-TRIM21 mRNA only or with mCherry-TRIM21 mRNA and mouse anti-aPKC antibody (n = 45 cells from 6 embryos). d, Schematic representation of the TRIM-Away experiment. e, Immunofluorescence analysis of anti-mouse secondary antibody to detect the electroporated aPKC antibody (green), mCHERRY (red), and DAPI nuclear staining (blue) in cow morula stage embryos electroporated with mCherry-TRIM21 mRNA and mouse anti-aPKC antibody at a pulse length of either 5 or 7.5 msec (n = reported in Supplementary Table 10 for each condition). f, Immunofluorescence analysis of anti-mouse secondary antibody to detect the electroporated aPKC antibody (green), YAP1 (red), GATA3 (magenta) and DAPI nuclear staining (blue) at the morula stage in control embryos or embryos electroporated with mCherry-TRIM21 mRNA and mouse anti-aPKC antibody. g, h, Quantification of YAP1 (g) and GATA3 (h) fluorescence intensity, normalized to DAPI intensity, in outer cells in cow control embryos or embryos electroporated with mCherry-TRIM21 mRNA and mouse anti-aPKC antibody (n = 154 cells for YAP1 from 16 embryos, and n = 207 cells for GATA3 from 20 embryos). t-test, ns = not significant. Scale bars as displayed in figures. i, Proposed model for human early lineage specification. EPI, epiblast; PrE, primitive endoderm; TE, trophectoderm. E-CAD, E-CADHERIN; β-CAT, β-CATENIN.

Fig. 1. Transcriptional and protein expression differences between cells at the morula stage in human embryos

a, Violin plot showing log-transformed size-factor-normalized expression of GATA3 in human morula cells. n = 197 cells. Black line corresponds to the median. Red highlights cells with lowest GATA3 expression and in blue are cells with high GATA3 expression. b, Graph interference of population heterogeneity dimensionality reduction analysis of human morula cells. Single cells colored with the log-transformed size-factor-normalized expression of GATA3. c, ATAC-seq chromatin accessibility in human embryos at the morula stage compared to the 8-cell stage. Examples of transcription factors with a significant change in activity score (p < 0.05) are highlighted in purple in the morula and in cyan in the 8-cell stage. d, Time-course immunofluorescence analysis of GATA3 (green), F-ACTIN (red), YAP1 (magenta) and HOECHST-33342 nuclear staining (blue) in human embryos at pre-compaction (n = 5), late compaction (n = 5), morula (n = 10), expanded blastocyst (n = 4) stages. e, f, Quantification of YAP1 (e) and GATA3 (f) fluorescence intensity, normalized to HOECHST-33342 intensity, in either inner or outer cells in human morula embryos (n = 95 cells for YAP1 and n = 79 for GATA3 from 10 embryos). t-test for YAP1 distribution, ****p < 0.0001; Mann-Whitney U test for GATA3 distribution, ****p < 0.0001. Yellow arrowheads point to outer cells expressing YAP1 and GATA3, cyan arrows mark inner cells lacking detectable YAP1 and GATA3 expression. g, Scatter plots showing positive correlation of GATA3 and KRT18 expression profile in human morula cells. n = 197 cells. r = Pearson correlation coefficient. Values displayed as log-transformed size-factor-normalized counts. The black line corresponds to a linear regression model fitted to the data with 95% confidence bands. h, Immunofluorescence analysis of GATA3 (green), KRT18 (magenta) and DAPI nuclear staining (blue) in human morula stage embryos (n = 3). i, Genome browser view of the ATAC-seq signal at the KRT18 locus. High confidence peaks (FDR < 0.001) were used to identify transcription factor motifs. Representative binding motifs associated with the footprints are highlighted. The average expression of KRT18 in high GATA3-high and GATA3-low expressing cells at the morula is shown and the TPM units indicated. Scale bars, as displayed in figures.

Fig. 2. Apical expression of aPKC and AMOT in outer cells in human morula stage embryos, where SOX2 expression is retained.

a, Scatter plots showing negative correlation of GATA3 expression profile with DUXA and KLF17 expression profiles in human morula cells. n = cells considered. r = Pearson correlation coefficient. Values are displayed as log-transformed size-factor-normalized counts. The black line corresponds to a linear regression model fitted to the data with 95% confidence bands. Scale bars, as displayed in figures. b, Immunofluorescence analysis of SOX2 (green), β-CATENIN (red), GATA3 (magenta) and DAPI nuclear staining (blue) in human embryos at the morula (n = 6) or expanded blastocyst (n = 5) stages. Yellow arrowheads point to outer cells expressing SOX2 and GATA3. c, Immunofluorescence analysis of aPKC (green), β-CATENIN (red), AMOT (magenta) and DAPI nuclear staining (blue) in human morula stage embryos (n = 10). d, Fluorescence intensity profile of aPKC and AMOT shown along the yellow arrows in human morula stage embryos. Scale bars, as displayed in figures.

Fig. 3. aPKC activity is required for YAP1 and GATA3 expression in mouse, cow and human morula stage embryos.

a, Immunofluorescence analysis of GATA3 (green), E-CADHERIN (red), YAP1 (magenta) and DAPI nuclear staining (blue) in control and aPKC inhibitor-treated mouse morula stage embryos. b, c, Quantification of YAP1 (b) and GATA3 (c) fluorescence intensity, normalized to DAPI intensity, in outer cells in control and aPKC-inhibitor treated mouse morula stage embryos (n = 243 cells for YAP1 from 28 embryos, and n = 191 for GATA3 from 26 embryos). t-test, ****p < 0.0001. d, Immunofluorescence analysis of GATA3 (green), β-CATENIN (red), YAP1 (magenta) and DAPI nuclear staining (blue) in control and aPKC inhibitor-treated cow morula stage embryos. e, f, Quantification of YAP1 (e) and GATA3 (f) fluorescence intensity, normalized to DAPI intensity, in outer cells in control and aPKC-inhibitor treated cow morula stage embryos (n = 209 cells for YAP1 from 19 embryos, and n = 218 cells for GATA3 from 21 embryos). Mann-Whitney U test, ****p < 0.0001. g, Immunofluorescence analysis of GATA3 (green), β-CATENIN (red), YAP1 (magenta) and DAPI nuclear staining (blue) in control and aPKC inhibitor-treated human morula stage embryos. h, i, Quantification of YAP1 (h) and GATA3 (i) fluorescence intensity, normalized to DAPI intensity, in outer cells in control and aPKC-inhibitor treated human morula stage embryos (n = 406 cells for YAP1 from 37 embryos, and n = 218 cells for GATA3 from 21 embryos). Mann-Whitney U test, ****p < 0.0001. j, Immunofluorescence analysis of GATA3 (green), YAP1 (red), anti-mouse secondary antibody to detect the electroporated aPKC antibody (magenta) and DAPI nuclear staining (blue) at the morula stage in embryos electroporated either with mCherry-TRIM21 mRNA only or with mCherry-TRIM21 mRNA and mouse anti-aPKC antibody. k, l, Quantification of YAP1 (k) and GATA3 (l) fluorescence intensity, normalized to DAPI intensity, in outer cells at the morula stage in embryos electroporated either with mCherry-TRIM21 mRNA only or with mCherry-TRIM21 mRNA and mouse anti-aPKC antibody (n = 281 cells for YAP1 from 32 embryos and n = 263 cells for GATA3 from 31 embryos). t-test, **p < 0.01, ****p < 0.0001. m, Immunofluorescence analysis of GATA3 (green), YAP1 (red), anti-mouse secondary antibody to detect the electroporated aPKC antibody (magenta) and DAPI nuclear staining (blue) at the morula stage in human control embryos and embryos electroporated with mCherry-TRIM21 mRNA and mouse anti-aPKC antibody. Yellow arrowheads point to decrease YAP1 and GATA3 expression in the TRIM-Away experiment. 2 independent experiments. n, o, Quantification of YAP1 (n) and GATA3 (o) fluorescence intensity, normalized to DAPI intensity, in outer cells at the morula stage in human control embryos and embryos electroporated with mCherry-TRIM21 mRNA and mouse anti-aPKC antibody (n = 88 cells from 8 embryos for YAP1 and n = 116 cells from 11 embryos for GATA3). Mann-Whitney U test, ****p < 0.0001. Scale bars as displayed in figures.