DEV028415 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Foxa2 and T mark different epiblast progenitors and descendents. (A) Mid-sagittal confocal sections of a pre-streak T^GFP/+ knock-in embryo showing mutually exclusive synthesis of GFP (green) and Foxa2 (red) protein in posterior epiblast cells. (B) At LS stage, the T^GFP/+ embryo shows distinct epiblast domains of GFP-positive (green arrow) and Foxa2-positive (red arrow) cells, which give rise to GFP-positive posterior mesodermal cells and double-positive anterior PS derivatives. Note that at this stage the GFP and Foxa2 expression in the epiblast overlap. DE, definitive endoderm; VE, visceral endoderm; PS, primitive streak.

• Supplemental Figure S2 -

  Fig. S2. T⁺ mesoderm cells and Foxa2⁺ endoderm cells show distinct morphology. (A-I) Mid-sagittal confocal sections of an MS-stage embryo showing brightfield (A) and whole-mount immunofluorescent staining of brachyury (T, red) or Foxa2 (green) with DAPI (blue) (B-E) or E-cadherin (E-cad, red) (F-I). Anterior is to the left and distal at the bottom. (A) The anatomical primitive streak (PS) is outlined by black dots. (B) Overlay of T, Foxa2 and DAPI staining. Note that Foxa2-positive epiblast (green asterisks) and DE cells are found anterior to the anatomical border of the PS. (C-D) Mesodermal cells double positive for Foxa2 and T are found at the end of the PS (yellow arrowhead), whereas flattened Foxa2-positive cells are found in the anterior PS, where they form a two-cell-layer thick row with the visceral endoderm (white arrows). (F) Along the proximal distal axis on the posterior side of the embryo the cells are subdivided according to their synthesis of T (red bar), Foxa2 and T (yellow bar) and Foxa2 (green bar) protein. (G) Areas for quantification of T-positive (top) and Foxa2-positive (bottom) cells. The circumference of the cells outlined by the E-cad staining was used to determine the length-width ratio of round T-positive (H) and flat Foxa2-positive cells (I). (J) Quantification of length-width ratio of Foxa2-positive (l=9.5±2.05 µm; w=4.4±1.08 µm; l/w=2.27±0.67; n=34) and T-positive (l=8.1±1.29 µm; w=5.0±1.25 µm; l/w=1.68±0.39; n=44) cells in three different embryos (P<0.02).

• Movie 1 -

  Movie 1. Definitive endoderm cells are morphogenetically distinct from mesoderm cells prior to intercalation into the visceral endoderm. Time-lapse analysis of a mid-to-late streak 4n dsRed ↔ wt YFP chimera. A mid-sagittal section of the distal proportion of the embryo (PS to the right) was imaged over 126 minutes at 3-minute intervals. Flattened green endoderm progenitor cells line the visceral endoderm (white arrows) and intercalate into the outer layer of visceral endoderm (red) over time. One cell moves anteriorly along the visceral endoderm before it intercalates into the outer epithelium (white asterisk). Mesoderm cells with round, mesenchymal morphology (red arrows) extend filopodia towards, but remain segregated from, the outer visceral endoderm layer.

• Movie 2 -

  Movie 2. Foxa2-deficient ‘endoderm-like’ cells show elongated morphology but do not stably integrate into the overlying visceral endoderm. Time-lapse imaging of definitive endoderm formation in a MS stage 2n dsRed ↔ Foxa2^−/− YFP chimera. A sagittal confocal section (distal tip of the embryo at the bottom and PS to the right) was imaged for 126 minutes at 3-minute intervals. Green Foxa2 mutant endoderm-like cells with elongated morphology (white arrows) line the visceral endoderm layer. Over time these cells round up, lose contact with the visceral endoderm layer and end close to the epiblast. On the top right, EMT of a Foxa2 mutant cell can be observed.

• Movie 3 -

  Movie 3. Foxa2 mutant cells transiently intercalate into the visceral endoderm but fail to epithelialize. Time-lapse imaging of a late-streak 4n dsRed ↔ Foxa2^−/− YFP chimera. Sagittal confocal section (distal tip of the embryo at the bottom, PS to the right) imaged for 99 minutes with a time interval of 3 minutes. A Foxa2-deficient endoderm-like cell has intercalated into the visceral endoderm (white asterisk). However, this cell fails to epithelialize and rounds up, leaves the outer cell layer and continues to migrate anteriorly. Elongated Foxa2 mutant endoderm-like cells lining the visceral endoderm fail to intercalate and migrate quickly out of the focal plane. At the end of the movie a continuous layer of visceral endoderm (indicated in blue) covers the chimeric embryo.

• Movie 4 -

  Movie 4. Polarization of endoderm cells during intercalation into the epithelial layer. Time-lapse imaging of an intercalating endoderm cell in a mid-streak 4n YVI ↔ wt Lyn-Tomato chimera. Sagittal confocal sections at the posterior side of the embryo were imaged for 57 minutes at 3-minute intervals. Over 20 time points, the cell was followed through three optical sections 8 µm apart. At the beginning of the intercalation process, a non-polarized endoderm cell (circumference of this cell indicated by white dots) extends filopodial processes into the outside visceral endoderm. During intercalation, the Lyn-Tomato protein becomes concentrated at the leading edge of the migrating cell and results in an epithelial cell integrated into the outer visceral endoderm with Lyn-Tomato protein highly concentrated on its apical membrane.