Journal: Cell Reports

Article Title: Integrin β1 coordinates survival and morphogenesis of the embryonic lineage upon implantation and pluripotency transition

doi: 10.1016/j.celrep.2021.108834

Figure Lengend Snippet: Integrin β1 is necessary for epiblast cell survival upon implantation and pluripotency transition (A–C) Analysis of apoptosis by cleaved caspase-3 staining in wild-type (fl/fl, top) and mutant (Δ/Δ, bottom) mESC spheroids at 24 (A), 48 (B), and 72 h (C) of culture. Steady increase in the number of c-caspase-3 + structures in mutants over time. Fisher’s exact test: (A) p = ns (number of spheroids n = 42 [fl/fl], n = 44 [Δ/Δ]); (B) ∗∗∗∗ p < 0.0001 (n = 38 [fl/fl], n = 48 [Δ/Δ]); and (C) ∗∗∗∗ p < 0.0001 (n = 26 [fl/fl], n = 28 [Δ/Δ]). (D–G) In vivo -recovered post-implantation embryos at E5.5, as shown in Figure S1 D: epiblast is either fl/fl or Δ/Δ; visceral endoderm (VE) and extraembryonic ectoderm (ExE) are +/+. In contrast to wild-type embryos (Epi:fl/fl) (D), embryos deficient for integrin β1 (Epi:Δ/Δ) either have a small epiblast (E), show few residual Oct4 + cells (F), or lack epiblast completely. Quantification of the number of embryos with epiblast versus degenerated/lethal—Fisher’s exact test: ∗∗∗ p = 0.0003 (number of embryos n = 15 [fl/fl], n = 25 [Δ/Δ], 4 replicates). (H–J) Assessment of apoptotic cells in chimeric blastocysts at E4.5–4.75, Epi:fl/fl (H, top) or Epi:Δ/Δ (H, bottom): embryos are sequentially re-stained to assess NANOG cells in the c-caspase-3 channel. Mutant embryos (Epi:Δ/Δ) contain a significantly higher number of apoptotic cells within the epiblast compartment: quantification of the percentage of c-caspase-3 + cells over total epiblast cell number (I), and percentage of apoptotic cells in wild type (median = 1.7%) versus mutants (median = 14%), Mann-Whitney test; ∗∗∗∗ p < 0.0001 (number of embryos n = 37 [fl/fl], n = 29 [Δ/Δ]). Correlation between the percentage of c-caspase-3 + cells with the percentage of either OTX2 (top) or NANOG + cells (bottom) within the epiblast (J): exit from naive pluripotency (increase in OTX2 and decrease in NANOG cells) correlates with the increase in the percentage of c-caspase-3 + cells (number of embryos n = 23 [fl/fl], n = 15 [Δ/Δ]). (K and L) Wild-type and mutant mESCs display comparable minimal levels of apoptotic cells when cultured in 2iLIF (naive pluripotency status, absence of OTX2 expression). (K) Fisher’s exact test: p = ns (number of spheroids n = 33 [fl/fl], n = 37 [Δ/Δ]). In the absence of 2iLIF (L), cells transit to formative pluripotency as shown by the expression of OTX2 and activate the apoptotic pathway in the absence of integrin β1 (Δ/Δ). Scale bars: 5 μm (A), 10 μm (B, C, K, and L), 15 μm (H, inset), 25 μm (H, inset, and D–F).

Article Snippet: Integrin β1 (for human embryos) , Merck Millipore , MABT821.

Techniques: Staining, Mutagenesis, In Vivo, MANN-WHITNEY, Cell Culture, Expressing

Journal: Cell Reports

Article Title: Integrin β1 coordinates survival and morphogenesis of the embryonic lineage upon implantation and pluripotency transition

doi: 10.1016/j.celrep.2021.108834

Figure Lengend Snippet: Fine-tune spatial segregation between integrin β1 and actomyosin (A) Configuration of the mouse epiblast before implantation (E4.5): cells are apolar and display heterogenous localization of integrin β1 (Itg-β1) and actomyosin, as shown by the distribution of phalloidin (F-actin) and phosphorylated non-muscle myosin (pMLC-II). (B) Configuration of the mouse epiblast upon implantation (E5.0): segregation in a mutually exclusive manner between integrin β1 and actomyosin while cells acquire a wedge-shape morphology. (C) Configuration of the mouse epiblast at post-implantation (E5.5): cells of the mature epiblast epithelium are columnar in shape surrounding a central pro-amniotic cavity. Integrin β1 localizes on the basolateral domain and actomyosin on the apical side. (D) Integrin β1 and actomyosin are heterogeneously distributed in mouse embryonic stem cells (mESCs) maintained in naive pluripotency (+2iLIF, left panel), similarly to E4.5. Integrin β1 and actomyosin become spatially segregated during formative pluripotency following 72 h of removal of 2iLIF (−2iLIF, right panel), similarly to E5.5. Scale bars: 25 μm (A–D). Magnified/inset areas indicated by arrows (A and B).

Article Snippet: Integrin β1 (for human embryos) , Merck Millipore , MABT821.

Techniques:

Journal: Cell Reports

Article Title: Integrin β1 coordinates survival and morphogenesis of the embryonic lineage upon implantation and pluripotency transition

doi: 10.1016/j.celrep.2021.108834

Figure Lengend Snippet: Loss of integrin β1 causes basal actomyosin accumulation and morphogenesis failure (A and B) Distribution of actomyosin in mESC after 24 (A) and 48 h of culture (B). Wild-type cells (fl/fl) display enrichment of F-actin and p-myosin at the central point of the apical membrane initiation site (AMIS) at 24 h (A, top). Apical actomyosin is maintained after 48 h at the center of the rosette configuration (B, top). Mutant cells (Δ/Δ) display ectopic accumulation of actomyosin basally (A, bottom), giving rise to a large basal actomyosin cable after 48 h of culture (B, bottom). (C) Blebs and ectopic E-cadherin junctions appear on the basal domain of mutant mESCs (arrows). (D) Distribution of actomyosin in the mouse blastocyst at E4.5, chimeric for either fl/fl (top) or Δ/Δ (bottom) alleles of Itgβ1 . Wild-type epiblasts (Epi:fl/fl) accumulate actomyosin at the central point of apical constriction, forming a rosette configuration (top). Mutant epiblasts (Epi:Δ/Δ) accumulate actomyosin basally and fail to form the rosette (bottom). (E) Fluorescence intensity quantification of apicobasal p-myosin (pMLC-II) at E4.5 shows significant increase of basal myosin in mutant epiblasts. Fluorescence intensity = mean ± SEM, Mann-Whitney test: ∗∗∗∗ p < 0.0001 (number of embryos n = 21 [fl/fl], n = 23 [Δ/Δ]). (F) Assessment of actomyosin localization in post-implantation embryos at E5.5. Wild-type epiblasts (Epi:fl/fl) accumulate actomyosin at the apical side of the epiblast epithelium. Mutant epiblasts (Epi:Δ/Δ) accumulate actomyosin basally and fail to form the central pro-amniotic cavity. (G) Fluorescence intensity quantification of apicobasal p-myosin (pMLC-II) at E5.5: significant increase in basal myosin in mutant epiblasts. Fluorescence intensity = mean ± SEM, Mann-Whitney test: ∗∗∗∗ p < 0.0001 (number of embryos n = 14 [fl/fl], n = 8 [Δ/Δ]). (H) Quantification of percentage of embryos forming cavity in the epiblast at E5.5: all mutant epiblasts fail to form the cavity: Fisher’s exact test: ∗∗∗∗ p = 0.0001 (number of embryos n = 14 [fl/fl], n = 8 [Δ/Δ]). Scale bars: 5 μm (A), 10 μm (B and C), 25 μm (D), and 50 μm (F).

Article Snippet: Integrin β1 (for human embryos) , Merck Millipore , MABT821.

Techniques: Membrane, Mutagenesis, Fluorescence, MANN-WHITNEY

Journal: Cell Reports

Article Title: Integrin β1 coordinates survival and morphogenesis of the embryonic lineage upon implantation and pluripotency transition

doi: 10.1016/j.celrep.2021.108834

Figure Lengend Snippet: Integrin β1 is dispensable for the establishment but necessary for the maintenance of apicobasal polarity (A) Distribution of the polarity marker PAR3 in mESCs at 1- and 2-cell stage (24 h of culture in −2iLIF). Wild-type cells (fl/fl, top) recruit PAR3 toward the AMIS similarly to mutant cells (Δ/Δ, bottom). (B) Quantification of the orientation of apicobasal polarity at 24 h: Fisher’s exact test: p = ns (number of mESC spheroids n = 42 [fl/fl], n = 39 [Δ/Δ]). (C and D) Distribution of centrosomes, as shown by γ-tubulin staining, at 2-cell stage and quantification of the angles along the nuclear-centrosome axis. Centrosome-nuclear axis angle: means ± SEMs (red). Test: Mann-Whitney test: p = ns (number of mESC spheroids n = 26 [fl/fl], n = 31 [Δ/Δ]). (E and F) Assessment of polarization in agarose at 24 h by Golgi and PAR3 localization. Fisher’s exact test: p = ns (number of spheroids n = 38 [fl/fl], 38 [Δ/Δ]). Both wild-type and mutant cells display correct apicobasal polarity at 24 h of culture, even in the absence of ECM components. (G) Assessment of polarization at 48 h. PAR6 is recruited at the apical site where actomyosin accumulated in wild type. PAR6 is recruited basally at the site of actomyosin accumulation in mutant cells. (H) In wild type, podocalyxin vesicles are recruited apically at PAR6 site. In mutant, PAR6 localizes basally, and podocalyxin vesicles are secreted basally at this latter site. (I) Quantification of the orientation of apicobasal polarity at 48 h, assessed by Golgi and PAR6 localization. Fisher’s exact test: ∗∗∗∗ p < 0.0001 (n = 30 [fl/fl], n = 29 [Δ/Δ]). Scale bars: 5 μm (A, C, and E) and 10 μm (G and H).

Article Snippet: Integrin β1 (for human embryos) , Merck Millipore , MABT821.

Techniques: Marker, Mutagenesis, Staining, MANN-WHITNEY

Journal: Cell Reports

Article Title: Integrin β1 coordinates survival and morphogenesis of the embryonic lineage upon implantation and pluripotency transition

doi: 10.1016/j.celrep.2021.108834

Figure Lengend Snippet: In vitro rescue of integrin-mediated survival and morphogenesis via FGF/IGF stimulation and ROCK inhibition (A) ROCK inhibition by Y27632 rescues lumen initiation in mESCs deficient for integrin β1 (Δ/Δ, bottom): Golgi is correctly oriented apically, podocalyxin vesicles are secreted apically, polarity marker aPKC is recruited at the apical domain, and actomyosin is re-established apically. Quantification of the percentage of mESC spheroids undergoing lumenogenesis shows no significant differences between wild-type and mutant cells: Fisher’s exact test: p = ns (number of spheroids n = 65 [fl/fl], n = 68 [Δ/Δ]). (B) Assessment of structures showing apoptotic cell death following ROCK inhibition treatment: mutant cells activate apoptosis despite rescue of polarity and lumen formation. Fisher’s exact test: ∗∗∗∗ p < 0.0001 (number of spheroids n = 35 [fl/fl], n = 39 [Δ/Δ]). (C) Stimulation of the survival pathways via supplementation of FGF2, IGF1, and GSK3i is not sufficient to prevent initiation of apoptosis in integrin β1 mutant cells (Δ/Δ): Fisher’s exact test: ∗∗∗ p = 0.0002 (n = number of spheroids = 35 [fl/fl], n = 35 [Δ/Δ]). (D) Inhibition of ROCK coupled to supplementation of FGF2, IGF1, and GSK3i restores both morphogenesis and survival in spheroids deficient for integrin β1 (Δ/Δ, bottom): Fisher’s exact test: p = ns (number of spheroids n = 47 [fl/fl], n = 40 [Δ/Δ]). (E and F) Fluorescence intensity quantification of apicobasal p-myosin (pMLC-II) in mESCs spheroids cultured for 48 h in medium only shows myosin ectopic localization on the basal side of mutant cells (ratio <1). Fluorescence intensity = mean ± SEM, Mann-Whitney test: ∗∗∗∗ p < 0.0001 (number of spheroids n = 31 [fl/fl], n = 30 [Δ/Δ]). (G and H) Fluorescence intensity quantification of apicobasal p-myosin (pMLC-II) in mESCs spheroids supplemented with FGF2, IGF1, GSK3i, and ROCKi for 48 h shows re-establishment of myosin in the apical domain in mutant cells (ratio >1). Fluorescence intensity = mean ± SEM, Mann-Whitney test: ∗∗ p = 0.002 (number of spheroids n = 35 [fl/fl], n = 31 [Δ/Δ]). Despite the increase in the ratio of apicobasal p-myosin, mutants still differ significantly from wild types. Scale bars: 10 μm (A–D, F, and H),

Article Snippet: Integrin β1 (for human embryos) , Merck Millipore , MABT821.

Techniques: In Vitro, Inhibition, Marker, Mutagenesis, Fluorescence, Cell Culture, MANN-WHITNEY

Journal: Cell Reports

Article Title: Integrin β1 coordinates survival and morphogenesis of the embryonic lineage upon implantation and pluripotency transition

doi: 10.1016/j.celrep.2021.108834

Figure Lengend Snippet: In vivo rescue of survival and morphogenesis in mouse embryos and spatial segregation of integrin β1 and actomyosin in the human embryo (A) Culture of mouse embryos from pre- to post-implantation as shown in Figure S3 F: wild-type controls (Epi:fl/fl) develop into post-implantation egg-cylinders (left), mutant epiblasts deficient for integrin β1 (Epi:Δ/Δ) fail to undergo lumenogenesis and to survive during post-implantation development in normal culture conditions (center). Supplementation with ROCKi, FGF2, IGF1, and GSK3i restores lumenogenesis and survival of the epiblast compartment in integrin β1-deficient embryos (right). (B) Quantification of the percentage of embryos undergoing lumenogenesis in wild-type and mutant embryos cultured in normal conditions compared to mutant embryos cultured in the presence of ROCKi, FGF2, IGF1, and GSK3i. Fisher’s exact test: fl/fl versus Δ/Δ ROCKi/FGF2/IGF1/GSK3i p = ns; Δ/Δ medium versus Δ/Δ ROCKi/FGF2/IGF1/GSK3i ∗∗∗∗ p < 0.0001 (number of embryos n = 26 [fl/fl], n = 16 [Δ/Δ], n = 51 [Δ/Δ, ROCKi/FGF2/IGF1/GSK3i]). (C) Schematic summary of results. Wild-type (left): integrin β1-mediated adhesion to the basement membrane leads to suppression of actomyosin basally, allowing its apical localization. Activation of actomyosin at the apical side leads to the apical localization of PAR6 ( Figure 4 G) and secretion of podocalyxin vesicles and initiation of lumenogenesis ( Figure 4 H). Integrin β1 promotes epiblast survival via stimulation of FGF/ERK and IGF1/AKT pathways. Mutant (right): loss of integrin β1 leads to ectopic actomyosin accumulation basally. The basal actomyosin recruits PAR6 basally, which directs podocalyxin vesicles toward the basal side, preventing lumenogenesis. Loss of integrin β1 leads to apoptosis. (D) Assessment of the localization of integrin β1 and actomyosin in human embryos at day 8 d.p.f. shows initiation of spatial segregation between the 2 complexes in a mutually exclusive manner (arrows). At 10 d.p.f., the 2 are fully segregated, with integrin β1 localizing on the basolateral domain, while actomyosin is confined on the apical side of the epiblast epithelium facing the central amniotic cavity. Scale bars: 25 μm (A) and 50 μm (D).

Article Snippet: Integrin β1 (for human embryos) , Merck Millipore , MABT821.

Techniques: In Vivo, Mutagenesis, Cell Culture, Membrane, Activation Assay

Journal: Cell Reports

Article Title: Integrin β1 coordinates survival and morphogenesis of the embryonic lineage upon implantation and pluripotency transition

doi: 10.1016/j.celrep.2021.108834

Figure Lengend Snippet:

Article Snippet: Integrin β1 (for human embryos) , Merck Millipore , MABT821.

Techniques: Recombinant, Software

Journal: Antibodies

Article Title: Tuning Relative Polypeptide Expression to Optimize Assembly, Yield and Downstream Processing of Bispecific Antibodies

doi: 10.3390/antib7030029

Figure Lengend Snippet: IgG expression of K2O30, K2O35, K2O41 and their respective codon de-optimized variants after transfection in Transformed Human Embryo Kidney monolayer epithelial cells (PEAK cells). ( A ) Electrophoresis using the Agilent Bioanalyzer 2100 for total IgG obtained after the first affinity chromatography step capturing all three IgG forms. The bands corresponding to the common heavy chain (HC), the lambda light chain (λ LC) and the kappa light chain (κ LC) in reducing and denaturing conditions are indicated. ( B ) Ratio of the light chains as determined from the band intensity of the Agilent result. ( C ) Total IgG concentration in the culture supernatant.

Article Snippet: Transformed Human Embryo Kidney monolayer epithelial cells (PEAK cells; Edge Bio, La Jolla, CA, USA) were maintained in 5% CO 2 at 37 °C in a humidified atmosphere in DMEM (Invitrogen, Carlsbad, CA, USA) containing 10% FCS (Sigma-Aldrich, St Louis, MO, USA) and supplemented with 2 mM glutamine (Sigma-Aldrich, St Louis, MO, USA), referring to complete DMEM.

Techniques: Expressing, Transfection, Transformation Assay, Electrophoresis, Affinity Chromatography, Concentration Assay