DEV006858 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. The specificity of morpholino activity. The specificity of the morpholino activity was checked by GFP immunohistochemistry after co-injection of the morpholino and a plasmid vector encoding the complementary sequence of the morpholino in front of the EGFP sequence. When this construct and MO1 were co-injected, the translation of EGFP was impaired. EGFP production was tested by immunohistochemistry with a specific antibody (Molecular Probes) and visualized with the ABC Kit (Pierce).

• Supplemental Figure S2 -

  Fig. S2. snail1b and no tail (ntl) expression domains. The posterior limit of snail1b expression in the axial mesendoderm coincides with the anterior limit of no tail (ntl) expression.

• Supplemental Figure S3 -

  Fig. S3. Wild-type and snail1b morphants at the end of gastrulation. (A) Wild type. (B) An embryo in which the hypoblast layer has reached the anterior position and formed the polster, but that has failed to complete epiboly. The prechordal plate was abnormally enlarged. (C) When embryos complete epiboly, the abnormal shape of the prechordal plate is more apparent.

• Movie 1 -

  Movie 1. Sagittal view of wild-type shield cells grafted into a wild-type host. Donor embryos were injected at the one- to two-cell stage with mRNA for a membrane tagged-RFP. Cells (10-20) were grafted at the shield stage into the shield of the donors and time-lapse movies were recorded over 30 minutes, taking z-stacks every 2 minutes. The movies were recorded from 70% epiboly and show the region posterior to the prechordal plate. Note the different protrusions and the dynamic contacts with neighbouring cells while migrating to the anterior part of the embryo (left).

• Movie 2 -

  Movie 2. Sagittal view of snail1b morphant shield cells grafted into a wild-type host. Donor embryos were injected at the one- to two-cell stage with a construct for a membrane form of RFP together with 3 ng of Snail1b morpholino. Cells (10-20) were grafted at the shield stage into the shield of the donors and time-lapse movies were recorded over 30 minutes, taking z-stacks every 2 minutes. The movies were recorded from 70% epiboly and show the region posterior to the prechordal plate. The grafted cells move as compact groups that maintain strong intercellular adhesion and a typical epithelial structure. Anterior is to the left.

• Movie 3 -

  Movie 3. Sagittal view of the anterior mesendoderm in a wild-type embryo. Time-lapse movie recorded over a period of 1 hour at 5 minute intervals, starting at 90% epiboly, of single parasagittal confocal sections. Anterior is to the left. Frame size: 250 μm×125 μm. Note the compaction of the migrating cells, the formation of the prechordal plate and the morphological boundary that separates it from the rest of the axial mesendoderm.

• Movie 4 -

  Movie 4. Sagittal view of the anterior mesendoderm in a snai1b morpholino-injected embryo. Time-lapse movie of single parasagittal confocal sections recorded over a period of 1 hour at 5 minute intervals, starting at 90% epiboly. Anterior is to the left. Frame size: 250 μm×125 μm. Note the poor compaction and the cells that lag behind, struggling to move.