Animal manipulation and experimental procedures were conducted according to the national and European Union legislation regarding the use of animals for experimental purposes, and under the license of the national regulatory agency (DGAV – Direção Geral de Alimentação e Veterinária) and Institutional Animal Care and Use Committee (CEBEA – Comissão de Ética e Bem-Estar Animal; Ref. 001/2018). Male and female Crl: CD1 (ICR) (CD1) mice were purchased from Charles River Laboratoire France and maintained at the Faculty of Veterinary Medicine of the University of Lisbon animal house facilities. Mice were maintained in a 12 h light/dark cycle, in corn cob bedded cages and with ad libitum access to standard laboratory diet and water. Mouse health was monitored daily.
Embryo collection and in vitro culture
Embryos were obtained from 2-3 months-old CD1 female mice, following superovulation and mating with CD1 males. Briefly, females were injected intraperitoneally with 10 IU equine chorionic gonadotropin (Intergonan; MSD Animal Health, Portugal) and 46 h later with 10 IU human chorionic gonadotropin (hCG; Chorulon; MSD Animal Health). Females were then housed overnight with a male and the presence of a vaginal plug was checked the following morning (0.5 dpc). At 2.5 dpc, females were euthanized by cervical dislocation under general anesthesia (intraperitoneal injection with 150 mg kg-1 ketamine + 10 mg kg-1 xylazine) and embryos were collected by oviduct flushing with M2 medium (Sigma-Aldrich, St Louis, MO, USA). Morphologically normal 8 to 16-cell embryos were selected, washed in M2 medium and in vitro cultured in groups of 20 in 500 μl of KSOM (Millipore, Specialty Media, Germany) overlaid with 400 μl of mineral oil (EmbryoMax®, Millipore), in 4-well dishes (Nunclon, Nunc, Roskilde, Denmark), at 37 ⁰C in a 90% N2 + 5% O2 + 5% CO2 humidified atmosphere. Following a 24, 36 and 48 h culture (corresponding to respectively 3.5 dpc, 4.0 dpc and 4.5 dpc), embryos were classified into the CM, BL, EBL, eHBL (early HBL) and HBL developmental stages, according to Nagy et al. (2003)  (Figure 6).
Gene transcription analysis - qRT-PCR
Quantification of transcripts of Notch components – receptors (Notch1, Notch2, Notch3 and Notch4), ligands (Delta-like1 - Dll1, Delta-like4 - Dll4, Jagged1 and Jagged2), and effectors (Hes1 and Hes2) – and of transcripts of pluripotency and differentiation gene markers Sox2, Klf4, Oct4, Cdx2, Cdca7 and Lgr5 was analyzed in individual 3.5 dpc CM (n= 9), BL (n= 9) and EBL (n= 7) and 4.5 dpc HBL (n= 5). Overall, transcription was individually evaluated in 30 embryos.
RNA extraction of single embryos was performed using the Arcturus® PicoPureTM RNA Isolation Kit (Applied Biosystems, ThermoFisher Scientific, USA) and DNA digestion with RNase-free DNase Set (Qiagen, Hilden, Germany). Concentration and purity of RNA were assessed spectrophotometrically at 260 and 280 nm (NanoDrop®2000c, ThermoFisher Scientific). Complimentary DNA (cDNA) synthesis was performed using Maxima First Strand cDNA Synthesis Kit for RT-qPCR (ThermoFisher Scientific) using 20 ng of total RNA in each reaction. Pre-amplification of cDNA was achieved with SSoAdvancedTM PreAmp Supermix (BioRad, CA, USA) using 10 μl of undiluted cDNA and a primer pool of genes Notch1-4, Dll1 and Dll4, Jagged1-2, Hes1-2, Sox2, Klf4, Oct4, Cdx2, Lgr5, and reference genes Rps29 and Hprt1 (Table 3). With the exception of Sox2, which is coded by a single exon, primers were designed to bracket two exons to avoid genomic DNA amplification. In the case of Sox2, the cDNA specific amplification was confirmed with a minus-reverse transcriptase control.
Pre-amplified cDNA was diluted 1:10 in Tris-EDTA buffer and kept at -20 ⁰C until qRT-PCR analysis. This was performed in duplicate wells in StepOne PlusTM (Applied Biosystems, ThermoFisher Scientific) in 96-well optical reaction plates (Applied Biosystems), using the universal temperature cycles: 10 min of pre-incubation at 95 ⁰C, followed by 40 two-temperature cycles (15 s at 95 ⁰C and 1 min at 60 ⁰C). Melting curves were acquired to ensure that a single product was amplified in the reaction. Each reaction used 10 μl of Perfecta® Sybr® Green Fast Mix, ROX™ (Quanta bio, MA, USA), 2 μl of diluted pre-amplified cDNA (corresponding to 0.2 ng of cDNA) and 80 nM of each primer in a total reaction volume of 20 μl. A NTC (no-template control) was included in all reaction plates and only plates with undetermined Ct in NTC wells were analyzed. Also, only wells with a single specific melting curve peak were analyzed. For each gene, one PCR product was run through a 2.5% agarose gel to confirm expected product size and the identity of this PCR product was confirmed by DNA sequencing. All reactions with the same Tm as the confirmed PCR product were considered specific. Positive controls were added to each reaction plate to exclude primer design artifacts: mouse uterus in oestrus for Notch1, Dll4 and Hes1 transcription, mouse uterus in metoestrus for Notch2, Notch3 and Hes2 transcription, mouse uterus in dioestrus for Notch4, Jagged1 and Jagged2 transcription , and mouse small intestine for Dll1 and Lgr5 . Embryos themselves were used as positive controls for Sox2, Klf4, Oct4, Cdx2 and Cdca7 transcription [18, 19].
The first step in transcription data analysis was the calculation of prevalence among embryos, i.e. the proportion of embryos with detected transcription at each developmental stage. Genes with a Ct value > 35 were considered without amplification. This was further confirmed by visualization of qRT-PCR products in agarose gels (Table 1 and Figure 1). The next step in transcription analysis was to quantify transcription levels of most prevalent genes. This was performed by two approaches. In Figure 2 panel A, Ct values were normalized to housekeeping gene 1 (Rps29) and the ∆Ct values obtained further calibrated with housekeeping gene 2 (Hprt1), generating ∆∆Ct values. These values were log transformed and results presented as the Log2 of power of ∆∆Ct values. The Log2 of power of ∆Ct values of transcription levels of housekeeping genes Rps29 and Hprt1 at each developmental stage are shown in Figure 2 panel B. The second approach is shown in Figure 2 panel C. Here, Ct values of each target gene were normalized with the mean Ct values of housekeeping genes Rps29 and Hprt1, and the obtained ∆Ct values were then calibrated to ∆Ct values of compact morulae (shown as 0.0), originating the ∆∆Ct values for log transformation . Results are also presented as the Log2 of power of ∆∆Ct values.
Gene expression analysis - immunocytochemistry
Embryos (3.5 dpc BL) were fixated in a 4% paraformaldehyde solution for 30 min, at 4 ⁰C, permeabilized in phosphate-buffered saline (PBS) + 0.5% Triton X-100 for 1 min and washed in PBS. Blocking was performed in a PBS + 0.1% Tween20 solution containing 2.5% bovine serum albumin (Sigma-Aldrich) for 1 h at room temperature, followed by a 4 ºC overnight incubation with the primary antibody diluted in blocking solution. Primary antibodies, all polyclonal and already validated for use in mouse cells [36, 47], were diluted as presented in Table 4. Negative IgG controls were performed using rabbit polyclonal IgG (ab27478, Abcam) and goat polyclonal IgG (ab37373, Abcam) at the appropriate dilutions. Embryos were then washed in PBS (4 × 5 min) and incubated with AlexaFluor® 594 chicken anti-rabbit (A11012, Life Technologies, USA) or chicken anti-goat (A21468, Life Technologies) secondary antibody diluted 1:300 in blocking solution, according to primary antibody host species, for 30 min, at room temperature. Embryos were then washed 2 × 10 min in PBS followed by Hoechst33268 (Sigma-Aldrich) nuclear labeling and finally mounted in ProLong™ Gold Antifade Mountant (Life Technologies). For each primary antibody, 6 blastocysts were analyzed, and a Z-stack was captured using a Zeiss LSM 710 confocal microscope (Carl Zeiss Microscopy, Oberkochen, Germany) with an optical magnification of 400× and treated with Fiji software (National Institutes of Health, USA).
Embryo culture supplementation with Notch ligands and a Notch signaling inhibitor
Mouse 8-16 cell embryos were collected and in vitro cultured as previously described, being randomly allocated in groups of 20 to each of the following treatment groups: i) Control, without treatment ii) Jagged1, medium supplemented with 1 μg ml-1 Jagged1 (1277-JG, R&D Systems, Bio-Techne, USA); iii) Jagged2, medium supplemented with 1 μg ml-1 Jagged2 (4748-JG, R&D Systems); and iv) DAPT, medium supplemented with 100 µM DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; Sigma-Aldrich). The experiment considered 10 in vitro culture sessions (550 embryos) until 3.5 dpc (24 h), from which 9 sessions (511 embryos) were further cultured until 4.0 dpc (36 h), and from the latter, 6 sessions (301 embryos) were further cultured until 4.5 dpc (48 h). Embryos were evaluated for viability, expressed as non-degenerated morphologically normal embryos progressing in culture, and their developmental stage recorded at those time-points (Figure 7) by a technician blinded to group assignment, according to criteria established by Nagy et al. (2003) . Five to six individual 4.0 dpc EBL from each group were processed for quantification of transcripts of Notch genes (Notch1-2, Jagged1-2, Hes1) and pluripotency and differentiation marker genes (Sox2, Klf4, Oct4, Cdx2, Cdca7), as described above.
Statistical analysis was performed using the statistical software SPSS Statistics (version 22, IBM® SPSS® Statistics, 2013, IBM, NY, USA). Real-time PCR data (ΔCt values) did not follow normal distribution (Figure S1) and were transformed to log 2 of power of ∆∆Ct for normalization, which allowed the use of parametric tests. Regarding Notch1, Notch2, Jagged1, Jagged2, Hes1, Sox2, Oct4, Klf4 and Cdx2 transcription, ANOVA was performed to compare the relative transcription between developmental stages, followed by LSD post-hoc analysis. Two-sided Pearson correlation coefficient was calculated to investigate the relationship between the transcription of Notch components, and between the latter and the transcription of pluripotency/differentiation markers. Chi-square test was used to evaluate the effect of Jagged1, Jagged2 and DAPT medium supplementation on in vitro cultured embryo viability and developmental rates. Results were considered significant if p < 0.05.