The timing of polarization at the 8-cell stage is asynchronous.

A) Schematic representing mouse pre-implantation development and live imaging of apical-basal polarization through the 8-cell stage, with the polarization marker EZRIN-RFP microinjected at the 2-cell stage into both blastomeres. B) Time-lapse images of mouse embryos through the 8-cell stage showing EZRIN-RFP fluorescence, with apical domains indicated by the white arrowhead, and times post-8-cell stage division on each image. Apical domains (see Materials and Methods for definition) form at different times at the 8-cell stage. An example of a cell classed as ‘late polarization’ (LP) time in the top image and ‘early polarization’ (EP) time in the bottom image. C) Plot of polarization times throughout the mouse 8-cell stage, indicating two distinct peaks of polarization (‘early’ – EP, ‘late’ – LP), n = 177 blastomeres counted and polarization defined in Methods. D) EZRIN-RFP accumulation at the cortical surface and the inter-blastomere contact angle measured over time from the start of the changes in inter-blastomere angle associated with compaction, for representative LP and EP blastomeres – see Materials and Methods for details on calculation. Scale Bar = 15 µm.

Early polarizing blastomeres accumulate PARD6 with F-ACTIN and have distinct morphological properties.

A) Immunostaining of early 8-cell stage embryo, with white pointers indicting blastomeres containing both PARD6 and F-ACTIN accumulation at the outer cell cortex (EP) and red pointer indicating a blastomere with neither. B) Smoothened quantification of PARD6 and F-ACTIN across the outer membrane of an EP cell (white pointer, lower image) and an unpolarized cell (red pointer, lower image) at the early 8-cell stage. C) Method for calculating the apical-nucleus distance and apical domain size of blastomeres at the late 8-cell stage after assessment of polarity status, from live imaging using EZRIN-RFP, see Materials and Methods for further details. D) EP cells have a significantly smaller apical-nucleus distance than LP cells, Mann-Whitney test, ****p<0.0001, n = 22 EP cells, n = 59 LP cells analysed. E) EP cells have a significantly larger apical domain size than LP cells, two-tailed t test, ****p<0.0001, n = 14 EP cells, n = 82 LP cells. Scale Bar = 15 µm

Early polarizing blastomeres show higher levels of CDX2 mRNA at the early 8-cell stage and CDX2 protein at the late 8-cell stage.

A) Projection of representative Z slices in the uncompacted embryo. Immunostaining discerns EP (early polarizing) from unpolarized cells. Hybridization chain reaction for CDX2 and for NANOG or TEAD4 (positive controls, expression of these mRNAs is higher than CDX2 at the 8-cell stage in all cells (Deng et al., 2014). Red arrowhead indicates EP cells and the window shows an amplified display of CDX2 puncta. B) Mean number of CDX2 puncta in early polarizing (EP) and unpolarized cells. C) Schematic showing reduced system of 8-cell stage doublets. EZRIN-RFP mRNA is injected into both blastomeres of the CDX2:GFP 2-cell embryo; blastomeres are separated at the 4-cell stage and imaged until the late 8-cell stage. D) Representative images of doublets with nuclear CDX2-GFP and EZRIN-RFP. Top images shows a doublet with two LP cells (‘late polarizing’), labelled as left and right, and the bottom shows a doublet with an EP and LP cell (‘early polarizing’). The white arrowhead indicated an EP cell. E) Normalized CDX2 intensity (to cytoplasmic background signal) over time in a representative doublet, ending at the first cell division that marks the final frame of the 8-cell stage. EP and LP cells are indicated. F) EP doublets have a significantly higher CDX2-GFP ratio when comparing the EP cell to the LP cell, than when comparing two LP cells in the LP doublets (Left to Right), Mann-Whitney test, n = 49 doublets total (7 EP), *p<0.05 Scale Bar = 15 µm.

Early polarizing blastomeres are biased towards symmetric cell divisions and the trophectoderm lineage.

A) Schematic indicating injection and time-lapse imaging to assess apical domain formation via EZRIN-RFP, and nuclear CDX2-GFP accumulation. B) Representative images showing time-lapse recordings of embryos over pre-implantation development during and after the 8-cell stage. Apical domain formation throughout the 8-cell stage is monitored by following EZRIN-RFP. Symmetric divisions are those in which both daughter cells inherit the EZRIN-RFP-positive apical domain after cell division, whereas in asymmetric divisions only one cell inherits the apical domain. Trophectoderm contribution can be assessed using the CDX2:GFP signal – outer, CDX2-GFP positive cells are counted as trophectoderm. C) EP cells have significantly greater frequency of symmetric divisions (left) and trophectoderm progeny (right). Two-tailed Fischer’s exact tests, ****p < 0.0001 and **p < 0.01 respectively; n = 199 LP cells and 46 EP cells were counted from, with 77 symmetric LP divisions (38.7%) and 33 EP symmetric divisions (71.7%); n = 275 LP cells and n = 87 EP cell progeny analyzed for lineage contribution at the 16-cell stage, with 188 LP trophectoderm cells (68.3%) and n = 72 EP trophectoderm cells (82.8%). Scale Bar = 15 μm.

Early polarizing blastomeres have increased blastomere width.

A) Indication of method for measuring the apex angle (degrees), side length and full length (μm) in the embryo mid-plane before cell division, using time-lapse images with EZRIN-RFP and LIFEACT-GFP markers. B) Blastomeres dividing symmetrically have a higher length ratio and larger apex angle than asymmetrically dividing blastomeres. n = 45 embryos with n = 100 asymmetrically dividing and n = 55 symmetrically dividing cells analyzed. Any embryos with ambiguous division pattern were excluded, two-tailed student’s t-test, ****p < 0.0001 in both tests. C) Blastomeres from B re-classified according to their polarity status, with 13 EP blastomeres and 155 LP blastomeres identified. Early polarizing blastomeres have a higher length ratio and larger apex angle than LP blastomeres, two-tailed t-test, **p < 0.01 and *p < 0.05 respectively. Any embryos with ambiguous polarity status were excluded. D) Snapshot of 3D analysis of 8-cell embryos using 3D Mesh Deformation Plugin on Fiji – see Method for further details. E) Example embryo with EP and LP cells indicated, with their aspect ratio over time. A higher aspect ratio indicates a higher ratio of long axis: short axis length. F) 32 cells from 4 embryos with at least 2 EP cells each were analyzed in 3D at the end of the 8-cell stage, and aspect ratio measured and normalized to the embryo average. EP cells have a significantly larger normalized aspect ratio than LP cells, two-tailed t-test, *p < 0.05. Scale Bar = 15 µm.

Frequency of early polarization is increased after inhibition of CARM1 activity.

A) Schematic indicating experimental design for CARM1 inhibition using CARM1(E267Q) or CARM1 inhibitor. B) Immunostaining for DAPI, HA-tag (tagged to the CARM1(E267Q) construct), and injected EZRIN-RFP (at the early 8-cell stage). EZRIN-RFP present in injected blastomeres and an EP blastomere is indicated via an arrow. C) CARM1(E267Q) blastomeres have a significantly higher frequency of being early polarized than Control (EZRIN-RFP only) injected blastomeres at the early 8-cell stage, N = 4 independent experiments, two-tailed z-test, **p < 0.01; overall n = 97 CARM1 (E267Q) embryos with n = 385 cells (73 EP, 19.0%), n = 100 Control embryos with n = 377 cells (37 EP, 9.8%). Mean of individual experiments with S.E.M given on the graph. D) CARM1i blastomeres have a significantly higher frequency of being early polarized than Control (EZRIN-RFP only) injected blastomeres at the early 8-cell stage, two-tailed z-test, ***p < 0.001; N = 2 independent experiments, overall n = 32 CARM1i embryos with n = 372 cells (40 EP, 11%), n = 47 Control embryos with n = 376 cells (20 EP, 5 %). Mean of individual experiments with S.E.M given on the graph. Scale Bar = 15 µm.

Frequency of early polarization is increased after BAF155 upregulation.

A) Schematic indicating experimental design for overexpression (OE) of BAF155 mRNA. B) BAF155 OE blastomeres have a significantly higher frequency of being early polarized than Control (EZRIN-RFP only) injected blastomeres at the early 8-cell stage, two-tailed z-test, *p < 0.05; N = 3 independent experiments, overall n = 35 BAF155 OE embryos with n = 140 cells (35 EP, 25.0%), n = 30 Control embryos with n = 120 cells (16 EP, 13.3%). Mean of individual experiments with S.E.M given on the graph. C) Immunostaining for DAPI, EZRIN and HA-tag (tagged to the BAF155 construct) at the early 8-cell stage. EZRIN-RFP present in injected blastomeres and an EP blastomere is indicated via an arrow. D) Schematic representing findings in this study integrating concepts from other studies. Blastomeres in the early embryo with low CARM1 activity (Torres-Padilla et al., 2007; Goolam et al., 2016)and elevated levels of its downstream target BAF155 (Panamarova et al., 2016) are biased towards polarizing early. Early polarization occurs through the canonical RhoA, TEAD4 and TFAP2C pathway (Zhu et al., 2017, 2020) and involves many of the same apical proteins, however these cells are distinguished by a number of factors which are consistent with its bias towards symmetric cell divisions and the trophectoderm lineage: early upregulation of Yap and activation of CDX2 (Jedrusik et al., 2008; Skamagki et al., 2013), an apical nucleus(Ajduk et al., 2014), wider geometry (Niwayama et al., 2019) and assembly of keratin filaments(Lim et al., 2020). Scale Bar = 15 µm.

Early polarization is not dependent on imaging or mating type, or embryo effects.

A) Representative images showing endogenous EZRIN-RFP signal in embryos injected with 330 ng/μl or 660 ng/μl. B) The concentration of injected mRNA does not affect the frequency of early polarizing (EP) cells, n = 82 blastomeres counted for 330 ng/μl from 11 embryos (7 EP cells), n = 75 blastomeres counted for 660 ng ng/μl from 10 embryos (7 EP cells), two-tailed z-proportion test, n.s.is non-significant, p > 0.05, N = 3 independent experiments. C) The frequency of early polarizing embryos does not differ significantly between time-lapse imaging and fixed embryos, two-tailed z-proportion test, n.s.is non-significant, p > 0.05, n= 87 embryos analyzed in time-lapse videos (19 EP), n = 32 fixed samples (10 EP). An EP embryo is one that contains at least one EP cell, as defined in Materials and Methods. Time-lapse imaging was conducted with microinjection of EZRIN-RFP into blastomeres, at the 2-cell stage, and embryos were imaged from the 8-cell stage, with blastomeres polarising in the first hour being counted as EP. For fixed embryos, Pard6 was used as the marker and embryos were fixed one hour after division to the 8-cell stage. Please see Materials and Methods for further details. D) Frequency of early polarizing embryos does not differ significantly between naturally mated and super-ovulated mice, n = 87 super-ovulated embryos (19 EP), n = 16 naturally-mated embryos (4 EP). two-tailed z-proportion test, n.s.is non-significant, p > 0.05. E) Representative immunostaining images (using PARD6 as a polarity marker) of embryos from super-ovulated CD1 and BL6SJL females at the 8-cell stage. F) Quantification of percentage of EP cells in different mouse strains, n = 56 CD1 blastomeres (7 embryos), 6 EP cells (10.7%); n = 120 BL6SJL blastomeres (15 embryos), 10 EP cells (8.3%) with comparison to previously reported quantification in F1 mice (7.0% EP cells). There is no significant difference in percentage of EP cells in any strain from all pairwise comparisons, two-tailed z-proportion tests, n.s.is non-significant, p > 0.05. G) Theoretical number of EP cells per embryo assuming independent cell autonomy, calculated by 8Cn X Pn X (1-P)8-n where p is the probability of an EP cell and n is the number of EP cells in the embryo, compared with measured values. Scale Bar = 15 µm.

Microtubule depolymerization does not affect the frequency of early polarization.

A) Schematic of disaggregating 4-cell stage embryos into individual blastomeres before treatment with colcemid or DMSO. B) ALPHA-TUBULIN and PARD6 immunostaining of early 8-cell doublets in control and colcemid-treated conditions. C) No significant difference between percentage of EP cells in control and colcemid-treated early 8-cell doublets, two-tailed t-test, p > 0.05, n = 49 colcemid-treated cells, n = 51 control-treated cells. Scale Bar = 15 µm.

Early polarization requires TEAD4, TFAP2C and RhoA

A) Schematic indicating experimental design to deplete TFAP2C and TEAD4 simultaneously. B) Depletion of TFAP2C and TEAD4 simultaneously by dsRNA injection causes elimination of EP cells, N = 2 independent experiments, n = 17 dsTead4+dsTfap2c embryos with n = 134 cells (0 EP), n = 22 Control embryos with n = 170 cells (8 EP), overall percentages given on graph, two-tailed z-test, *p < 0.05. C) Schematic representing treatment of embryos with C3 Transferase to inhibit RhoA, or with a control treatment. D) Immunostaining images showing RhoA inhibited embryos versus control embryos stained for DAPI and PARD6, indicating binucleation of RhoA embryos and arrows pointing to EP cells. E) Inhibition of RhoA causes elimination of EP cells, N = 3 independent experiments, n = 8 RhoAi embryos with n = 32 binucleated cells (0 EP), n = 16 Control embryos with n = 125 cells (11 EP), overall percentages given on graph, each binucleated cell counted as two cells for statistical analysis, two-tailed z-test, *p < 0.05. Scale Bar = 15 µm.

Nuclear YAP, but not nuclear CDX2, is higher in EP versus unpolarized blastomeres of uncompacted embryos.

A) Immunostaining of uncompacted, early 8-cell stage embryos for YAP and PARD6. EP cells are indicated with an arrowhead and have apical PARD6 accumulation. B) Immunostaining of uncompacted, early 8-cell stage embryos for CDX2 and PARD6. EP cells are indicated with an arrowhead and have apical Pard6 accumulation. C) The nuclear:cytoplasmic ratio of YAP in EP and unpolarized blastomeres; two-tailed t-test, ****p < 0.0001; N = 4 independent experiments, n = 16 embryos, with n = 124 cells (23 EP). D) Ratio of average nuclear CDX2 in EP versus unpolarized blastomeres across different embryos. n = 12 EP embryos. Scale Bar = 15 µm.

Early polarization is not dependent on division timing or order.

A) schematic showing experimental design to measure division and polarization timing. B) No significant difference between EP and LP populations in timing of third cleavage, n = 47 cells assessed, two-sample t-test, n.s. – not significant p>0.05. C) Order of division is not correlated with frequency of EP cells, n = 22 cells assessed, histogram correlation computed, n.s. – not significant, p>0.05.

CARM1 (E267Q) and CARM1 inhibitor verification using H3R26me2 staining.

A) Immunostaining for H3R26me2 (double methylation of arginine 26 on histone H3) in early 8-cell embryos injected with CARM1(E267Q). B) Quantification of nuclear H3R26me2 (normalized to background cytoplasm) shows significantly lower H3R26me2 nuclear intensity in CARM1(E267Q) blastomeres than Control blastomeres, two-sample t-test, **p<0.05, n = 10 Control blastomeres from 3 embryos and n = 15 CARM1(E267Q) blastomeres from 4 embryos. C) Immunostaining for H3R26me2 (double methylation of arginine 26 on histone H3) in early 8-cell embryos treated with CARM1 inhibitor or just vehicle. D) Quantification of nuclear H3R26me2 (normalized to background cytoplasm) between inhibitor and control treatments for 8 cell stage cells, showing a significantly higher concentration for the control treatment compared with CARM1 inhibitor, two-tailed t test, ****p < 0.0001, n = 3 inhibitor treated embryos (22 cells analyzed), n = 2 control treated embryos (14 cells analyzed). All graphs shown with standard error of the mean. Scale Bar = 15 µm.

KRT18 mRNA is upregulated in early polarized versus unpolarized blastomeres.

A) Representative z-slice of immunostaining of early 8-cell stage, late 8-cell stage and E3.5 embryos for KRT18. B) Graph showing the frequency of KRT18-positive cells at different stages during early pre-implantation murine development. We found that 6.77% of cells at the late 8-cell stage show keratin accumulation. C) Schematic of workflow: early 8-cell embryos were disaggregated into individual EP and unpolarized blastomeres before single cell RNA sequencing. Polarity status was determined by using live imaging of EZRIN-RFP. 10 EP and 10 unpolarized cells sequenced D) Expression levels of BAF155 and KRT18 determined by single-cell sequencing, two-tailed t-test, p = 0.0808 for BAF155 and p = 0.0394, *<0.05 for Krt18. Scale Bar = 15 µm.