DEV016402 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Premature activation of xOct91 and xID2 in xDMO morphants. RT-PCR analysis shows mis-expression of xOct91 and xID2 in xDMO morphants compared with wild-type controls. The maternal transcript xOct60 is a loading control.

• Supplemental Figure S2 -

  Fig. S2. Developmental expression analysis of genes upregulated in xDMO stage 8 embryos and developmental abundance of H3K4me3. (A) RT-PCR analysis of developmental gene expression patterns of xDMO gene targets. A variety of transcript types were observed: for example, xOct25 has a maternal component in eggs; whereas xOct91, xOct25 and xSox17β are zygotically expressed in a developmental window around gastrualtion/neurulation. By contrast, xCycD1 and xBF2 are essentially zygotic transcripts that persist in post-MBT embryos at subsequent stages of development. (B) Western blotting of staged Xenopus laevis histone-enriched extracts. Extracts were harvested as described in the Materials and methods before (St8) and after (St 11, 16, 24) MBT. Note the accumulation of H3K4me3 at St11, which is the equivalent of the mid-gastrulation stage and coincides with active zygotic gene transcription. By contrast, the mark is absent in xDMO8 histone isolations (bottom panel), a situation in which global gene activation occurs.

• Supplemental Figure S3 -

  Fig. S3. Silencing can be re-imposed in stage 8 xDMO morphants by wild-type and mutant hDNMT1 and quantitation by densitometry. (A) Whole-mount RNA in situ analysis of wild type (WT), xDMO morphants (xDMO), xDMO morphants plus wild-type hDNMT1 (wt) and xDMO morphants plus mutant hDNMT1 (mut). Stage 8 embryos were hybridised with xOct25 and xBF2 probes. Animal pole view are shown. Co-injection of either hDNMT1 (wt) or mutant hDNMT1 (mut) with xDMO decreases the level of ectopic expression of xOct25 or xBF2 compared with xDMO only. (B) Statistical analysis of whole-mount in situ rescue experiments. Densitometry was used (Aida Image Analyser) to quantitate hybridisation signals in four independent embryo sets. Standard errors were calculated using Microsoft Excel. Scale bar: 1 mm (S3A).

• Supplemental Figure S4 -

  Fig. S4. xBra is not activated in xDMO stage 8 morphants. Whole-mount RNA in situ analysis of wild-type (WT), xDMO morphants (xDMO) and antisense depleted (AS) stage 8 embryos. Stage 8 embryos were hybridised with xBra and xId2 probes. Animal pole views are shown. Notice xId2 is mis-expressed in AS and xDMO embryos, whereas xBra is misexpressed only in AS embryos. Global DNA hypomethylation only occurs in AS embryos (Stancheva and Meehan, 2000; Stancheva et al., 2001; Stancheva et al., 2002). Scale bar: 1 mm.