Fig 1.
Localization of E-cad and P-cad mRNA, and E-cad and Slug proteins, in HH stage 4–7 embryos.
(A-C, A’-C’) E-cad (CDH1) mRNA expression in HH stage 4, 5 and 7 embryos (A-C) and in transverse sections (A’-C’). (D-F, D’-F’) P-Cad (CDH3) mRNA expression in embryos at stages 4, 5 and 6 (D-F) and in transverse sections (D’-F’). E-cad mRNAs are downregulated in the pre ingression epiblast and in the primitive streak, while P-cad mRNAs persist in the primitive streak and medial mesoderm (arrows). (G-I) Immunofluorescence localization of Slug (green) and cadherin proteins using an antibody that recognizes E-cad and P-cad (red) in transverse sections through the middle streak region of HH stage 4, 5 and 7 embryos.
Fig 2.
Co localization of E-cad and P-cad in using a P-cad-specific antibody prepared against a peptide from the extracellular domain of P-cad that shares only three amino acids with the corresponding sequence in E-cad (A), and a commercially obtained antibody raised against the E-cad intracellular domain that recognizes both P-cad and E-cad. (B-D) Transverse section through the mid streak region of a HH stage 4 embryos, showing immunolocalization E-cad and/or P-cad (B), or P-cad (C), or both (D).
Fig 3.
Protein localization in transverse sections of gastrulating chicken and mouse embryos.
(A-C) Localization of E-cad and/or P-cad, p120-catenin, GM130 and ZO1 in transverse sections of HH stage 4 embryos. (A, B) The same microscopic field showing expression of E-cad and/or P-cad (A) and p120-catenin (B). The proteins colocalize at the periphery of cells in the epiblast, primitive streak and mesoderm. (C) Colocalization of E-cad and/or P-cad, GM130 and ZO1in a transverse section of a HH stage 4 embryo. Cells moving from the epiblast to the mesoderm retain E-cad and/or P-cad at their periphery while exhibiting a reorientation of Golgi that is characteristic of the change in cell polarity associated with EMT. (D) Transverse section through a E7.5 mouse embryo at the level of the primitive streak, visualizing E-cad and/or P-cad proteins. (E) Higher magnification of the boxed area in (D), showing persistence of E-cad and/or P-cad proteins on the surface of cells in the mesoderm below and near the primitive streak (arrow). Asterisks are rounded cells in the mesoderm that retain E-cad and/or P-cad protein.
Fig 4.
Localization of E-cad and/or P-cad and N-cad in transverse sections of a HH stage 3–4 chicken embryos.
(A) Brightfield image of a HH stage 3 embryo following processing for immunofluorescence localization of E-cad and/or P-cad and N-cad, showing the location of images in (B-D’). Expression of E-cad and/or P-cad (B,C,D), or E-cad and/or P-cad plus N-cad (B’, C’, D’) in transverse sections. (E, F) Higher magnification images of boxed area in (D’), showing the localization of E-cad and/or P-cad (E) and N-cad (F) as cells transition from epiblast to mesoderm. E-cad and/or P-cad are retained on the surface of cells during EMT and after cells emerge into the mesoderm. Arrows point to rounded cells in the ventral streak showing robust detection of E-cad and/or P-cad at the cell periphery. (G, H) Co localization of E-cad and/or P-cad and N-cad in transverse sections of HH stage 4 embryos, showing their heterogeneous expression between mesoderm cells.
Fig 5.
E-cad overexpression in gastrulating cells.
(A-B) Transverse sections of the primitive streak region of embryos electroporated with a myc tagged E-cad expression construct, visualizing in (A) myc (green) and Slug (white), and in (B) myc (green) and N-cad (red). Expression of E-cad had no effect on the ability of cells to undergo EMT and migrate into the mesoderm (arrows in B).
Fig 6.
Effects of SNAI2 morpholino on Slug protein expression and cell location.
(A) Reduction of Slug protein in most cells electroporated with morpholino targeting the initiation of translation of the Slug (Snail2) mRNA compared to the control (five base pair mismatch) morpholino. While most cells containing the control morpholino show high levels of Slug protein eight hours following electroporation as assayed by immunofluorescence, almost all cells containing the SNAI2 morpholino showed reduced or undetectable levels of Slug. (B) Relative location of epiblast cells electroporated with control or SNAI2 morpholino eight hours following electroporation, and the percent of cells at each location that are Slug-positive. Numbers of cells are shown above each bar. Whereas the percent of cells containing control morpholinos that are Slug-positive is similar at all locations, a higher percentage of Slug-positive cells containing the SNAI2 morpholinos were observed in the mesoderm away from the streak, indicating that SNAI2 morpholino containing cells lacking detectable Slug expression were preferentially located near the primitive streak.
Fig 7.
Location of SNAI2 morpholino and control morpholino containing cells in transverse sections of embryos.
A fluorescein-labeled morpholino targeting the initiation of translation of the SNAI2 mRNA, or a five base pair mismatch control morpholino, were electroporated into the epiblast of HH stage 3–4 embryos. (A-B) The same microscopic field visualizing cells containing the control morpholino (green) and Slug (red) in (A), or Slug (B). Almost all cells containing the control morpholino also express Slug protein (white arrows). (C-D) The same microscopic field visualizing cells containing the SNAI2 morpholino and Slug (C), or Slug (D). Most cells in the primitive streak containing SNAI2 morpholinos were Slug negative (white arrows). (E,F) Higher magnification views showing cells containing the SNAI2 morpholino, and lacking detectable Slug protein, undergoing EMT (arrows).
Fig 8.
SNAIL over expression is insufficient to induct EMT in the epiblast.
Transverse sections of embryos following electroporation of the epiblast with plasmids expressing a FLAG-tagged degradation resistant form of human Snail (6SAhSNAI1; A-E), myc tagged Rho-inhibitor peptide C3 (F, G, I, J) or pBEGFP (H). (A-C) Eight hours following electroporation with 6SAhSNAI1, no 6SAhSNAI1 expressing cells were observed in the mesoderm or exiting the epiblast. 6SAhSNAI1-expressing epiblast cells showed levels of E-cad and/or P-cad similar to untransfected cells. (D, E) Expression of 6SAhSNAI1 failed to induce basal lamina breakdown. (F,G) The same microscopic showing the epiblast and mesoderm of an embryo 4 hours following electroporation of the epiblast with the C3 expression construct. Virtually all C3-expressing cells (green) are observed in the mesoderm or exiting the epiblast (arrows). Some C3 positive cells were observed above the epiblast. (H) All cells expressing GFP remained in the epiblast. (I, J) Expression of C3 in epiblast cells induced basal lamina breakdown.
Fig 9.
C3 induces EMT without activating Slug or upregulating N-cad.
(A,B) Transverse sections showing the same microscopic field four hours following electroporation with the C3 expression vector. While cells expressing C3 (green) in the primitive streak region within the domain of normal Slug expression were also Slug-positive (arrowheads in A, B), C3 expressing cells in more lateral regions that were exiting the epiblast or that had migrated into the mesoderm failed to express detectable levels of Slug (arrows in A, B). (C, D) C3 expressing cells that had undergone EMT and migrated into the mesoderm failed to express N-cad.
Table 1.
EMT Induction by Snail and C3.