DEV02805 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Sna morphant phenotypes are rescued by injection of MASO-resistant mRNA. Sna morphants (middle image) show defects in PMC ingression as compared with controls (left). Right-hand image shows embryos of the same age as those in the middle image, co-injected with SnaMASO and ENG-Sna fusion mRNA (used as MASO-resistant mRNA), which exhibit normal gastrulation with rescue of ingression (n=30, 40% rescue). This experiment demonstrates the specificity of the SnaMASO used in this study.

• Supplemental Figure 2 -

  Fig. S2. Pigment cells are reduced in Sna morphants. (A,B) Control embryos show normal numbers of pigment cells (average 82.3±6.8). (C,D) Compared to the control, Sna morphants show significantly reduced pigment cells (average 40.2±5.0), and also stunted growth of anal arm rods.

• Supplemental Figure 3 -

  Fig. S3. Chimera experiments show that ingression of SMCs also requires Snail. (A-C) Experimental control (control micromeres combined with control host; n=13). Arrows show the SMC cells (pigment cells or blastocoelar cells). Control PMCs shown in green (B′). (C) Control chimera containing a normal number of pigment cells. (D-F) Chimera embryos (n=13) with control micromeres (in green, shown in E′) in a SnaMASO-host (in red, as an injection tag, not shown), which the SnaMASO is present in macromeres. The migrating SMCs are rarely observed at the tip of the archenteron of the chimera embryo (D), when compared with the control embryo (A). Fewer SMCs are seen in the blastocoel even at later stages (E,F). (G) Quantification of SMC numbers shows that Snail is involved in SMC ingression. At LG stage, any free mesenchyme cells that were not PMCs (not dyed green) were collectively counted, as these either could be blastocoelar cells or pigment cells (labeled as BC+PC). 1st and 2nd indicates two separate experiments (total n=13). At the 48 hour pluteus stage, the pigment cells are distinguishable as they appear in red, whereas blastocoelar cells could be counted also as cells free in the blastocoel and not green PMCs. The cell numbers of SMCs are significantly lower in these chimera embryos than in the controls.