|
Display additional annotations [+]
|
| |
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. |
|
Display additional annotations [+]
|
| |
Figure 1. TALEN-Induced Deletions Nullify Brachyury Function
(A) TALEN-induced 2- and 7-bp deletions in exon 1 of t (e1.2D) and exon 3 of t2 (e3.7D), and predicted frameshift translations generating truncated proteins of 59 and 170 amino acids (aa). These mutations were selected to generate a double heterozygous X. tropicalis line for the Brachyury paralogs t and t2 (te1.2D/+t2e3.7D/+).
(B) t and t2 transcript levels in hetero- and homozygous embryos as measured by qRT-PCR at early neurula stage (n = 3, mean ± SD). Two-tailed t test: ∗p ≤ 0.05.
(C) Multi-probe WMISH for various mesoderm cell lineage and derivative markers (actc1; cardiac and skeletal muscle; cav1, notochord; hoxd8, pronephros; myh6, heart; tal1, ventral blood island; tbx6, paraxial mesoderm) in wild-type and Brachyury (t/t2) null (KO) embryos, as well as embryos injected with four MOs targeting t and t2 (t/t2 MO mix) at mid-tailbud stage. Scale bar, 0.5 mm. |
|
Display additional annotations [+]
|
| |
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 |
