Research ArticleDEVELOPMENTAL BIOLOGY

Deciphering epiblast lumenogenesis reveals proamniotic cavity control of embryo growth and patterning

See allHide authors and affiliations

Science Advances  10 Mar 2021:
Vol. 7, no. 11, eabe1640
DOI: 10.1126/sciadv.abe1640
  • Fig. 1 Epiblast morphogenesis and initiation of lumenogenesis.

    (A) Blastocyst (E4.0-E4.5) and egg cylinder stage (E5.5) embryos stained for the basolateral marker Scrib and the apical marker Par6. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The Par6-positive domain facing the proamniotic cavity in the E5.5 embryo is indicated with a white arrowhead. (B) 3D culture of E14 ESC in N2B27 medium supplemented with 2i/Lif or Fgf2/Activin for 48 hours. The cells were stained for Scrib, Par6, and DAPI. The white arrowhead indicates the Par6-positive apical domain facing the central lumen. (C) E4.5-E5.5 embryos stained for Par6, E-cad, the epiblast marker Sox2, and DAPI. High-magnification images (bottom) show E-cad removal from the apical domain during proamniotic cavity formation (white arrowheads). (D) 3D culture of E14 ESC in N2B27 medium supplemented with Fgf2/Activin for 36, 48, or 72 hours. The cells were stained for Par6, E-cad, Sox2, and DAPI. The E-cad removal from the apical domain is indicated with white arrowheads. (E) Representative images from the time-lapse recording of ESCs expressing E-cad-tdTom grown in 3D culture conditions, in N2B27 medium supplemented with Fgf2/Activin, LY, and DNA live-stain dye Sir-DNA. Lumen initiation is detected by the accumulation of LY (red arrowheads) and decrease of E-cad-tdTom signal (white arrowheads). (F) Representative images from the time-lapse recording of ESCs expressing E-cad-tdTom grown in 3D culture conditions, in N2B27 medium supplemented with Fgf2/Activin, LY, and DNA live-stain dye Sir-DNA. Lumen initiation is detected by the accumulation of LY (red arrowheads) and decrease of E-cad-tdTom signal (white arrowheads). The paracellular fluid transport during mitosis is indicated with yellow arrowheads. Scale bar, 10 μm (A to F). Related to fig. S1 and movies S1 and S2.

  • Fig. 2 Reorganization of E-cad intercellular adhesion during lumenogenesis.

    (A) Schematic representation of the E-cad-WT-GFP and E-cad-LP-GFP constructs. Amino acid mutations in the juxtamembrane domain (JM) of E-cad-LP-GFP are shown. Amino acid sequences in red are required for ankyrin-G binding, and amino acid sequences in blue are required for clathrin-dependent endocytosis. ECD, ectodomain; TM, transmembrane domain; ICD, intracellular domain. (B) Schematic representation of trafficking of WT E-cad-WT-GFP (left) and the endocytosis-deficient mutant E-cad-LP-GFP (right). (C) ESCs expressing E-cad-WT-GFP or E-cad-LP-GFP cultured in N2B27 medium supplemented with Fgf2/Activin for 36, 48, or 72 hours. The cells were stained for GFP and Par6. Nuclei were counterstained with DAPI. (D) Quantification of the lumen volume from (C) based on Par6 staining, at least three independent experiments for each time point (36 hours, n > 25; 48 hours, n > 39; 72 hours, n > 72). Error bars represent SEM. P value was calculated using unpaired Student’s t test. *P <0.05; **P <0.01; ***P <0.001. (E) Schematic representation of the tetraploid complementation assay. (F) Live-microscopy images of egg cylinder stage embryos (n = 21) generated following tetraploid complementation using E-cad-WT-GFP–expressing ESCs. The emerging proamniotic cavity is marked with yellow arrowhead. (G) Live-microscopy images of egg cylinder stage embryos (n = 12) generated following tetraploid complementation using E-cad-LP-GFP–expressing ESCs. The apical localization of E-cad-LP-GFP is marked with white arrowheads. (H) Quantification of the lumen volume from (F) and (G) (E-cad-WT-GFP- E5.25, n = 15; E5.5, n = 4; E5.75, n = 2; E-cad-LP-GFP-E5.25, n = 5; E5.5, n = 5; E5.75, n = 2). Error bars represent SEM. Scale bar, 10 μm (C, F, and G).

  • Fig. 3 The CD34 family antiadhesins facilitates membrane separation.

    (A) E4.5-E5.5 embryos stained for E-cad, Podxl, and epiblast lineage marker Sox2. Nuclei were counterstained with DAPI. (B) 3D culture of E14 ESC in N2B27 medium supplemented with Fgf2/Activin for 36, 48, or 72 hours and stained for E-cad, Podxl, Sox2 and DAPI. (C) Reverse transcription polymerase chain reaction analysis of Podxl, Podxl2, and CD34 expression of E14 ESCs 3D cultured for 48 hours in N2B27 medium supplemented with 2i/Lif or Fgf2/Activin. (D) 3D culture of E14 ESC (control) and CD34 family triple-knockout ESC in N2B27 medium supplemented with Fgf2/Activin for 72 hours. The cells were stained for Podxl, Podxl2, and CD34, and Oct4 and DAPI. (E) 3D culture of E14 ESC (control) and CD34 family triple-knockout ESC in N2B27 medium supplemented with Fgf2/Activin. The cells were stained for Par6 and DAPI and 36, 48, or 72 hours of culture. (F) Quantification of lumen volume from (E) based on Par6 staining; at least three independent experiments (36 hours, n > 14; 48 hours, n > 73; 72 hours, n > 115). Error bars represent SEM. P value was calculated using one-way analysis of variance (ANOVA) with a Tukey’s post hoc test. **P < 0.01; ***P < 0.001. n.s., not significant. (G) Egg cylinder stage embryos (n = 10) generated following tetraploid complementation using control E14 ESC and stained for Podxl, Sox2, and DAPI. (H) Egg cylinder stage embryos (n = 15) generated following tetraploid complementation using CD34 family triple-knockout ESC and stained for Podxl, Sox2, and DAPI. Note that Podxl is expressed only in the extraembryonic lineages, the extraembryonic ectoderm and visceral endoderm, but not in the epiblast. Scale bar, 10 μm (A, B, D, E, G, and H). Related to fig. S2.

  • Fig. 4 Fluid influx via osmotic gradients initiates lumen formation.

    (A) E5.5 embryos (left) and 3D cultured ESCs stained for Podxl and Na+/K+-ATPase, CFTR, or Aqp4. (B) 3D culture of E14 ESCs in N2B27 supplemented with Fgf2/Activin. The CFTR inhibitor CFTR(inh)-172 (10 nM) and/or Na+/K+-ATPase inhibitor ouabain (100 μM), were added from 24 hours of culture. (C) Quantification of lumen volume from (B) based on Par6 staining; at least three independent experiments (36 hours, n > 6; 48 hours, n > 49; 72 hours, n > 48). Error bars represent SEM. P value was calculated using one-way ANOVA with a Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001. (D) 3D culture of CD34 family triple-knockout ESCs expressing E-cad-LP-GFP in the presence of CFTR inhibitor and/or Na+/K+-ATPase inhibitor. (E) Quantification of lumen volume from (D) based on Par6 staining; at least three independent experiments (36 hours, n > 11; 48 hours, n > 22; 72 hours, n > 54). Error bars represent SEM. P value was calculated using one-way ANOVA with a Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001. (F) Still images of the time-lapse microscopy of CD34 family triple-knockout cells expressing E-cad-LP-GFP. The site of lumen formation is indicated with a white arrowhead. (G) Still images of the time-lapse microscopy of CD34 family triple-knockout cells expressing E-cad-LP-GFP in the presence of the CFTR inhibitor and Na+/K+-ATPase inhibitor. Scale bar, 20 μm [A (embryo)] and 10 μm [A (ESCs), B, D, F, and G]. Related to fig. S3 and movies S3 to S5.

  • Fig. 5 Paracellular fluid transport mediates lumen expansion during mitosis.

    (A) Representative images from time-lapse microscopy of ESCs expressing membrane (E-cad-WT-tdTom) and nuclear (Histone H2B-tdTom) markers. The cells were grown in 3D culture for 72 hours in N2B27 medium supplemented with Fgf2/Activin in the presence of LY (cyan). The paracellular fluid flow during the subsequent stages of cell division is indicated with white arrowheads; a schematic of the process is represented on the lower panel. (B) Representative images from the time-lapse microscopy of egg cylinder stage embryos (E5.5) expressing membrane tdTomato. The paracellular transport of LY-marked fluid (cyan) to the proamniotic cavity is visible during cell division (white arrowheads). (C) Transmission electron microscopy images of the epiblast in E4.5 blastocyst, E5.5 egg cylinder, and 3D cultured ESCs for 48 hours. The paracellular spaces and the central cavity are marked with cyan (bottom); EPI, epiblast; VE, visceral endoderm. (D) Serial block-face scanning electron microscopy images of E14 ESCs grown in 3D culture conditions for 72 hours in N2B27 medium supplemented with Fgf2/Activin. Multiple intermembranous spaces (outline in cyan; magenta arrowheads) are visible around the central lumen. 3D volume rendering of the lumen and intermembranous spaces (outer surface, cyan; inner surface, gray). (E) Single plane of serial block-face scanning electron microscopy showing cells in telophase (magenta) and intermembranous pockets at the cleavage furrow (cyan). Scale bar, 10 μm (A and D); 20 μm (B); 10 and 5 μm [C (left and middle)] and 5 and 2 μm [C (right)]; and 5 μm (E). Related to fig. S4 and movies S6 to S8.

  • Fig. 6 The proamniotic cavity fluid is supplied by the blastocoel.

    (A) Whole-mount deciduae containing E4.75 and E5.5 embryos subjected to tissue-clearing procedure and stained for laminin and Podxl. Nuclei were counterstained with DAPI; the blastocoel is marked with a white line. The images are composed of several combined (projected) optical sections. The scattered laminin-positive cells at E5.5 are parietal endoderm cells of the enveloping Reichert’s membrane. (B) Schematic representation of blastocyst to egg cylinder growth and morphogenesis with blue arrows indicating the flow of blastocoel fluid into the emerging proamniotic cavity. The epiblast is marked with yellow, the extraembryonic ectoderm with white, and the primitive/visceral endoderm with gray; RM, Reichert’s membrane. (C) Still images of LY microinjection marking the blastocoel fluid in E4.5, E5.0, and E5.5 embryos. TE (white); RM (white), Reichert’s membrane; EPI (yellow), epiblast; PE (magenta); VE (magenta), visceral endoderm; ExE (red), extraembryonic ectoderm. A white arrowhead refers to the site of microinjection, and a yellow arrowhead refers to the proamniotic cavity. Scale bar, 10 μm (A).

  • Fig. 7 The proamniotic cavity is required for lineage communication and patterning of the early postimplantation embryo.

    (A) Schematic representation of the biotin-streptavidin interaction used to strengthen E-cad–mediated intercellular adhesion. (B) Western blot analysis of E-cad-bio-LP-tdTom biotinylation using streptavidin-HRP (horseradish peroxidase) antibody; the tdTomato tag was detected using anti-RFP antibody and used as a loading control. (C) 3D culture of ESCs expressing the E-cad biotin-streptavidin adhesion system. (D) Quantification of the lumen formation in (C) based on Par6 staining; at least three independent experiments (n > 132). Error bars represent SEM; P value was calculated using unpaired Student’s t test. ***P < 0.001. (E) Transmission electron microscopy of ESCs expressing the E-cad biotin-streptavidin adhesion system. (F) Embryos generated following tetraploid complementation using ESCs expressing the biotin-streptavidin adhesion system. Control embryos (n = 27) and embryos derived from foster mothers exposed to biotin (n = 12). (G) E5.5 control embryos (n = 15) and embryos derived from foster mothers exposed to biotin (n = 12) stained for Cer1, tdTomato, and DAPI. (H) E6.5 control embryos (n = 16) and embryos derived from foster mothers exposed to biotin (n = 13) stained for T-bry, tdTomato, and DAPI. (I) Live-microscopy images of egg cylinder stage embryo (E5.5-E6.0) generated following morula aggregation with ESCs expressing E-cad-WT-tdTom and GFP-Nodal and cultured in vitro to egg cylinder stage (n = 9). (J) Control embryos (p-Smad2 stained, n = 9; p-Smad1/5 stained, n = 7), embryos derived from biotin-treated foster mothers (p-Smad2 stained, n = 8; p-Smad1/5 stained, n = 11), and Epi-Nodal ko embryos (p-Smad2 stained, n = 11; p-Smad1/5 stained, n = 10). Scale bar, 10 μm [C, E (left), F, H, and I]; 5 μm [E (middle and right)]; and 20 μm (G and J). Related to fig. S5.

  • Fig. 8 Formation and function of the proamniotic cavity.

    Schematic representation of the proposed model of proamniotic cavity formation and function. AQP, Aquaporins.

Supplementary Materials

Stay Connected to Science Advances

Navigate This Article