DEV018853 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Ectopic and elevated WNT signalling activity in the Dkk1^−/− mutant embryo. (A-J) Expression of TOPGal reporter in (A-E) wild-type and (F-J) Dkk1^−/− embryos at E7.0 (A,F), E7.5 (B,G), E7.75 (C,H), E8.5 (D,I) and E9.5 (E,J) embryos. (F,G) Ectopic lacZ expression is detected in cells scattered in the anterior region (red asterisk; 1/5 E7.0 and 2/5 E7.5 embryos) and stronger expression in the posterior tissues (green asterisk, 4/5 E7.0 and 4/5 E7.5 embryos) of the Dkk1^−/− embryos. (H) By the late-bud stage (E7.75) stage, TOPGal expression was confined to the primitive streak of the Dkk1-null embryo as in the wild-type embryo, except for an apparently stronger expression in the tissues around the enlarged node with an exaggerated ventral indentation (bold red asterisk). In the Dkk1-null mutant, TOPGal-negative tissues were absent from the forebrain region of (I) the early-somite (n=6, E8.5) embryo and the truncated head of (J) the E9.5 (n=2) embryos. (K-P) Distribution pattern of β-catenin in the endoderm of (K,L) the anterior-proximal (AP, see inset), (M,N) the anterior-distal (AD, see inset) and (O,P) the posterior (PO, see inset) regions of E7.0 (K,M,O) wild type and (L,N,P) Dkk1^−/− embryos. Inset in K shows the regions (AD, anterior distal; AP, anterior proximal; PO, posterior) of the endoderm that were examined. Nuclei in (K,L,O,P) are stained by propidium iodide (red). In the mutant embryo, overall level of β-catenin staining is stronger than the wild-type counterpart and β-catenin is localised more frequently in the nuclei of the anterior endoderm cells (white arrows in L,N). Scale bar: 20 µm.

• Supplemental Figure S2 -

  Fig. S2. The lineage potential of Dkk1^−/− primitive streak cells. (A) Distribution of graft-derived cells in the host embryo. Data in the Dkk1^+/+ and Dkk1^−/− columns are presented as sets of three values: total cell scored (number of specimens) and percent of total cell population. Empty cells (somite, presomitc mesoderm, allantois)mean no graft-derived cells were found in the tissue. Dkk1^−/− cells can contribute to the anterior endoderm and mesoderm but not the paraxial and posterior mesoderm (red box). (B,C) Distribution of the EGFP-expressing Dkk1^−/− cells in the anterior tissues (B) 24 hours and (C) 36 hours after grafting of cells to the anterior primitive streak of the E7.0 wild-type host embryo (inset: 3 hours after grafting). (D) Whole-mount (left) and histological section (right) showing lacZ-positive graft-derived cells (arrows) in the heart and gut endoderm.

• Supplemental Figure S3 -

  Fig. 3. The pattern of displacement of endoderm cells in the Dkk1^−/− embryo during gastrulation. Electroporation of the distal endoderm of the (A) wild-type embryo and (B) Dkk1-null embryo 3 hours after electroporation (the insets) and after 24 hours of in vitro culture showing the localisation of EGFP-expressing endoderm cells in the embryo. Cells are spread more widely in the anterior and middle regions in the wild-type embryo, whereas cells are clustered in the anterior region in the mutant. Anterior side of the embryo is towards the left. (C-G) The distribution of EGFP-expressing endoderm cells after electroporation at (C) anterior-proximal (P<0.05), (D) anterior-distal (P<0.05), (E) distal (P<0.01), (F) posterior distal (P<0.02) and (G) posterior-proximal (P>0.05) sites of the embryo after 24 hours of in vitro culture (statistical significance tested by χ2-test). AYS and PYS: anterior and posterior yolk sacs. A,M,P: endoderm in the (A) anterior, (M) middle and (P) posterior third regions (A) of the early-somite stage embryo. Histograms show the distribution of the labelled cells (% of total population) in wild-type (wt) (purple bars) and Dkk1-null (−/−) embryos (yellow bars). (H,I) F-actin staining of the anterior endoderm cells of E7.0 (H) wild-type and (I) Dkk1^−/− embryo, showing the smaller apical surface area of cells in the mutant embryo. Scale bar: 10 µm.

• Supplemental Figure S4 -

  Fig. 4. Head morphology of E9.5 wild-type and Dkk1^+/−;Wnt3^+/− mutant embryos. Coronal sections were taken at the planes indicated by broken lines through the (A) branchial arches (BA) and (B) otic vesicle (OV). The mutant embryo (bottom row) displayed an open neural tube and a reduced foregut, representing a severe compound heterozygous phenotype.