DEV02149 Supplementary Material

Files in this Data Supplement:

• Supplemental Table - Adobe PDF

• Supplemental Figure 1 -

  Fig. S1. Wnt3a and Nodal are expressed in adjacent node cells. Two-color whole-mount in situ hybridization showing Wnt3a (orange) and Nodal (purple) expression in E7.5-8.5 embryos; (A-C,E,G) lateral views of left side of embryo, anterior is towards the left; (D,F,H) ventral views of the embryos depicted above, posterior end points up. (A) E7.5 early alantoic bud stage; Wnt3a transcripts were first detected in primitive streak ectoderm, prior to node formation, as Nodal expression is downregulated in embryonic mesoderm and distally restricted to the presumptive node. (B) E7.5 alantoic bud stage, (C,D) E7.75 neural plate stage. Wnt3a expression in the ectoderm overlaps Nodal expression in the underlying posterior ventral node endoderm. (E,F) E8.2, four-somite stage; Nodal is expressed asymmetrically in the node and in the left lateral plate mesoderm (LPM), while Wnt3a continues to be symmetrically expressed in the streak. (G,H) E8.5, eight-somite stage; Nodal expression is undetectable in the left LPM but remains asymmetric in the node, while Wnt3a expression persists in the streak. N, node; ps, primitive streak; a, allantois; lpm, lateral plate mesoderm.

• Supplemental Figure 2 -

  Fig. S2. Heart looping defects are not due to abnormal cardiac specification. Reduced Wnt/bcatenin signaling is associated with heart formation (Lickert et al., 2002; Marvin et al., 2001). To rule out the possibility that the heart-looping defects observed in the Wnt3a mutants were secondary to defects in cardiogenesis, we examined the expression of the heart markers Cripto, and Nkx2.5, the latter of which is required for proper looping morphogenesis (Lyons et al., 1995; Tanaka et al., 1999). Whole-mount in situ hybridization was performed on wild-type (A,C) and Wnt3a^-/- littermates (B,D) to examine Nkx2.5 expression in the heart at E8.75 (A,B), and Cripto expression in the outflow tract at the seven-somite stage (C,D). Both genes continued to be strongly expressed in hearts undergoing aberrant looping morphogenesis. The mutant Cripto-positive outflow tract remained in the midline at stages (E8.5) when it would normally have undergone a rightward shift in wild-type embryos. The broken white line outlines the looping ventricles. ot, outflow tract.

• Supplemental Figure 3 -

  Fig. S3. Node and axial markers are expressed in Wnt3a mutants. Whole-mount in situ hybridization of E8.2-8.5 wild-type (left) and mutant (right) embryos, analyzed for marker gene expression. Markers of the node and axial mesendoderm, such as Shh (A-D) and Foxa2 (E,F), continued to be expressed in three- to four-somite stage Wnt3a^-/- embryos (B,D,F). (G-J) Despite the loss of Lefty1 and Lefty2 (orange), normally expressed in the midline PFP and left LPM (G,I), Wnt11 (purple) continued to be expressed in the mutant node (arrow), posterior primitive streak and heart (H,J) as it was in wild-type embryos (G,I). (K,L) Two-color whole-mount in situ hybridization analysis of cryptic (purple) and Nodal (orange) expression. Cryptic was expressed in the midline, including the node, and bilaterally in the LPM of wild-type embryos (left embryos). Cryptic expression in the LPM and node was upregulated in six- to seven-somite stage Wnt3a^-/- embryos, suggesting that Wnt3a, directly or indirectly, represses cryptic transcription. We have previously shown that brachyury is also expressed in the Wnt3a^-/- node and notochord at these stages (Yamaguchi et al., 1999). (C,D,L) Ventral posterior view; (I,J) anterior view; the rest are lateral views; h, heart.

• Supplemental Figure 4 -

  Fig. S4. TOPgal expression from E7.5-8.5. The TOPgal reporter indicates that the canonical Wnt/b-catenin signaling pathway is active in the primitive streak at E7.5, when Wnt3a is first expressed there (A). Expression is easily detected in the node at E7.75 (B), persisting in the node and streak through E8.5 stages (C). Expression is also detected in the mid-hindbrain and heart tube. Staining was not observed in axial derivatives of the node such as the notochord or PFP.