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Display additional annotations [+]
Gene |
Clone |
Species |
Stages |
Anatomy |
chrd.1
|
|
laevis
|
NF stage 10
|
ectoderm
,
dorsal marginal zone
|
sox17a
|
|
laevis
|
NF stage 10
|
endoderm
,
blastopore
,
vegetal endoderm
|
cer1
|
|
laevis
|
NF stage 10
|
endoderm
,
endomesoderm
|
gsc
|
|
laevis
|
NF stage 10
|
mesoderm
,
dorsal marginal zone
|
hhex
|
|
laevis
|
NF stage 10
|
endoderm
,
endomesoderm
|
myf5
|
|
laevis
|
NF stage 10
|
mesoderm
,
marginal zone
,
dorso-lateral marginal zone
|
foxi1
|
|
laevis
|
NF stage 10
|
ectoderm
,
epidermis
,
animal hemisphere
|
krt12.4
|
|
laevis
|
NF stage 10
|
ectoderm
,
epidermis
,
animal hemisphere
|
|
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Fig. 2. H3.3/H3-depleted Xenopus embryos fail to express mesodermal marker genes. (A-I′) Control and injected embryos fixed at early gastrula were subjected to RNA in situ hybridization for the analysis of mesodermal (A-C′), endodermal (D-F′) and ectodermal (G-I′) marker gene expression. Representative embryos from three experiments are shown. (A-D,G) Vegetal views; (E,F) lateral views of bisected embryos, dorsal towards the right; (H) lateral view; (I) animal view. (J) Expression of selected genes in control and H3 MO-injected embryos at stage 10.5 was measured by qRT-PCR. All values were normalized to ornithine decarboxylase (ODC) and plotted relative to the respective transcript levels in control embryos. Error bars indicate s.d. of three independent experiments. *P<0.05 using a two-tailed Student t-test. |
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Fig. 4. Mesodermal gene activation is independent of post-translational modifications on H3.3 and incorporation at promoters. (A) Enrichment of H3.3-HA at the promoters of Xbra, siamois and cebpa was examined by ChIP-qPCR analysis of embryos injected with 750 pg of mouse H3.3a-HA mRNA at blastula (stage 8), early- (stage 10), mid- (stage 11) and late- (stage 12.5) gastrula stages. Level of enrichment is determined as a percentage of input. Expression of these genes at different developmental stages was measured by qRT-PCR. All values were normalized to ornithine decarboxylase (ODC) and plotted relative to the respective transcript levels in two-cell stage embryos. Error bars indicate s.d. of three independent experiments. (B) Rescue experiments were performed by co-injecting H3.3 MO with mRNA encoding HA-epitope tagged wild-type or mutant H3.3, H3.1 or H2Az. Injected embryos were either allowed to develop to late gastrula stage 12.5 (top panels), or fixed at stage 10.5 and subjected to whole-mount in situ hybridization analysis of Xbra expression (bottom panels). |
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Display additional annotations [+]
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Fig. S5. Mesoderm formation and gene expression in H3 MOs morphants is rescued by co-injection of mouse H3f3a mRNA. (A) Control and injected embryos fixed at early gastrula were subjected to RNA in situ hybridization analysis of Xbra and gsc transcripts. (B) Percentage of embryos co-injected with mRNA encoding HA-tagged wild-type or mutant H3.3, or H3.1 that arrested at early (stage 10.5-11) or late (stage 11.5-12) gastrula stages. (C) Rescue experiments were performed by co-injecting H3.3 MO with a total amount of 750 pg of mRNA encoding HA-tagged H3.1 or H3.3, or
both. Injected embryos were either allowed to develop to late gastrula stage 12.5 (top panels), or fixed at stage 10.5 and subjected to whole-mount in situ hybridization analysis of Xbra expression (bottom panels). (D) Percentage of injected embryos that arrested at early (stage 10.5-11) or late (stage 11.5-12) gastrula stages. |