JEB01666 Supplementary Material

Files in this Data Supplement:

• Supplemental Table 1 (PDF) - .
• Supplemental Table 2 (PDF)
• Supplemental Table 3 (PDF)
• Supplemental Table 4 (PDF)
• Supplemental Table 5 (PDF)
• Supplemental Table 6 (PDF) -

  Tables S1-S6. Contributions of labelled cells to specific regions of all blastocysts scored following injection of dsPar3 RNA, mRNA for aPKC, or control molecules.

• Supplemental Figure 1 -

  Fig. S1. Confirmation of effective downregulation by RNAi. (A) To confirm the specificity and effectiveness of RNAi, zygotes were injected with dsRNA against Par3 or GFP, cultured to the 2-cell stage and pooled into groups of 10 for the extraction of RNA. RT-PCR was carried out to detect either Par3 or β-actin RNA. Pooled embryos injected with dsGFP RNA (G) showed expression of both Par3 and β-actin (lanes 1 and 3), whereas pooled embryos injected with dsPar3 RNA showed no expression of Par3 and continued expression of β-actin (lanes 2 and 4). (B-E) Embryos were injected at the 4-cell stage with either a mixture of dsRNA against GFP and mRNA for DsRed (B,C) or with mRNA for DsRed alone ((D-E). The merged images (left hand panels) show the position of the labelled clone in red. The right hand panels show green fluorescence alone that is diminished in the embryo injected with dsGFP RNA (C). This is seen in the merged channel as a loss of the combined (yellow) signal in B, compared with its persistence in D.

• Supplemental Figure 2 -

  Fig. S2. Immunolocalization of aPKC in embryos injected with either wild-type (A-C) or dominant-negative aPKC (D-F) or DsRed (G-I). All embryos were co-injected with mRNA for DsRed as a lineage marker. Cells labelled with DsRed (A,D,G) also show cytoplasmic localization of aPKC (asterisk; B,E,H), whereas endogenous membrane localized aPKC (arrowheads) is seen in both labelled and unlabelled cells. Note that the increase in aPKC staining is not observed in control embryos (injected with DsRed only, G). DsRed forms dense aggregates (arrows) that are only visible through the red channel (C,F,I). Thus, cytoplasmic fluorescence of labelled cells through the green channel (B,E) is not due to DsRed. DNA is stained blue with TOTO-3 in the merged images (A,D,G). Bar, 10 mm.

• Supplemental Figure 3 -

  Fig. S3. Downregulation of E-cadherin prevents cell compaction. Individual blastomeres were co-injected at the 2-cell stage with E-cadherin dsRNA and synthetic mRNA for GFP. They were then examined at the 16-cell to 32-cell stage to reveal E-cadherin by immunofluorescence (red) and GFP (green). Left hand panels (A,C) show red channel alone; right hand panels (B,D) show both channels. It can be seen that cells expressing GFP have not compacted and have reduced levels of E-cadherin at their borders.