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Figure 3. Ubiquitous Immune Response against MO Intensifies during Embryogenesis
(A) Panel of genes upregulated in control and t/t2 morphants associated with the immune response and genes downregulated in t/t2 morphants and null mutants representing the Brachyury-dependent core network. Heatmap to the right represents the binding map of Brachyury (t) in the proximity (±40 kb) of indicated transcription start sites (TSS) at early tailbud stage (Gentsch et al., 2013).
(B) WMISH of immune response related gene transcripts c3ar1, tp53inp1, and tp53 in uninjected (uni) embryos and embryos injected with 18 ng of cMO or t/t2 MO mix. Left bottom corner inset, dorsal view of tailbud head showing elevated transcript levels in the CNS. tp53 antisense probe did not discriminate active isoforms shown in D. Scale bar, 0.5 mm.
(C) Temporal dynamics of transcript fold changes (log2 scale) for a selected group of genes representing the Brachyury-directed core network (tbx6) and the immune response (c3ar1, tp53inp1, and tp53) in MO-injected versus uninjected embryos as measured by qRT-PCR (n = 3, mean ± SD). Two-tailed t test (≥1.5-fold change): ∗p ≤ 0.1; ∗∗p ≤ 0.01; and ∗∗∗p ≤ 0.001.
(D) Genome map of full length tp53 and δ99tp53 transcript isoforms shows normalized transcript levels for uninjected (uni), control morphants (cMO), t/t2 morphants (t/t2 MO), wild-type (WT), t/t2 heterozygous (t/t2 het), and homozygous (t/t2 KO) mutant embryos at tailbud stages 26 and 34. Isoform-corresponding translation products with critical domains are on display below the heatmap: TAD, transactivation domain; DBD, DNA binding domain; NLS, nuclear localization signal; and OD, oligomerization domain. |
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Figure S3. Increased Transcription of tp53 Depending on GC Content of MO
Does Not Cause More Apoptosis, Related to Figure 3 and 4
(A) TUNEL assay on morphants and sibling embryos from double heterozygous t
-/+t2+/-
parents. DNase-treated wild-type embryos were used as positive controls. (B) Single
WMISH for tp53 and multi-probe WMISH for various mesoderm cell lineage and
derivative markers (cav1, notochord; hoxd8, pronephros; myh6, heart; tal1, ventral
blood island; tbx6, paraxial mesoderm) of late tailbud embryos injected with single
MOs or tracer sulforhodamine-dextran. Scale bar, 0.5 mm |
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Figure S5. Temperature and MO Dosage Effects on Brachyury Phenotype and
Immune Response Related Gene Transcription, Related to Figure 7
(A) WMISH for tp53 of late tailbud embryos injected with 4.5 or 18 ng of the t/t2 MO
mix and developed at 22 ºC or 28.5ºC. *, Remark: Increasing incubation temperature
also slightly up-regulated tp53 in the absence of any MO (confirmed by RT-qPCR,
data not shown). (B) Multi-probe WMISH for various mesoderm cell lineage and
derivative markers (cav1, notochord; hoxd8, pronephros; myh6, heart; tal1, ventral
blood island; tbx6, paraxial mesoderm) and single WMISH for tp53, tp53inp1 and
c3ar1 of mid-tailbud (stage 26) and late tailbud embryos (stage 34) injected with 6 or
18 ng of the t/t2 MO mix. White arrowheads point to the expression domains of tbx6
and cav1 that were not maintained in embryos without functional Brachyury. (C)
WMISH for tp53inp1 on wild-type (or t/t2 heterozygous) and t/t2 null mutant embryos
as well as embryos injected with 1 and 3 ng MO. The embryos were developed to
late tailbud stage 34 at 25-26ºC. Scale bar, 0.5 mm. |
