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Fig. 2. KDM3A is required for the primary
neurogenesis in Xenopus. (A) A schematic
showing the experimental design for B and C.
(B,B′) Embryos at stage 24/25 stained with red-Gal
and in situ hybridized with tubb2b. cMO or 3A MO
(40 ng) was injected into one cell at the two-cell
stage. The 3A MO-resistant kdm3a mRNA (300 pg)
together with 200 pg β-gal mRNAwas subsequently
injected into the 3A MO-injected cell at the two-cell
stage. The expression levels of tubb2b were
classified into three categories: ‘normal’, as seen in
cMO-injected embryos; ‘partial’, as seen in some of
KDM3A depleted embryos; and ‘complete’ loss, as
seen in the remainder of the KDM3A-depleted
embryos. Injection of the MO-resistant kdm3a
mRNA partially rescues the expression of tubb2b in
KDM3A morphants. The numbers on the top of
histograms in B′ are sums from two independent
experiments. (C,C′) Embryos at stage 16/17 stained
with red-Gal and in situ hybridized with tubb2b. cMO
and 3A MO (40 ng) were injected into one cell at the
two-cell stage. mRNA (100 pg) encoding neurog2
or ascl1 together with 200 pg β-gal mRNA was
subsequently injected into the MO-receiving cell at
the two-cell stage. Phenotypes were classified into
four categories based on whether ectopic neurons
were induced and/or whether tubb2b was ‘partially’
or ‘completely’ lost in the MO-injected side. The
numbers on the top of histograms in C′ are the total
number of samples from two independent
experiments. |
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Fig. 6. KDM3A-mediated demethylation of
H3K9me2 enhances Neurog2 recruitment
at the neurod1 promoter. (A) Western blot
detection of soluble histone H3, H3K9me1,
H3K9me2 and H3K9me3 in control and 2 ng
kdm3a mRNA-injected embryos. (B) Embryos
stained with red-Gal and in situ hybridized with
neurod1 (left panel, stage 18) or tubb2b (right
panel, stage 15). β-gal (200 pg) together with
1 ng kdm3a was injected into one cell at the
two-cell stage. (C) Western blot detection of
chromatin histone H3, H3K9me1, H3K9me2
and H3K9me3 in embryos without injection or
injected with 80 ng cMO or 3A MO. See
Materials and Methods for isolation of
chromatin histones. (D) Anti-H3K9me2 ChIPqPCR
analyses showing that 80 ng KDM3A
MO increased the H3K9me2 marks on the
neurod1 promoter. (E) Anti-H3K9me2 ChIPqPCR
analyses showing that Neurog2 was
unable to reduce the H3K9me2 marks on the
neurod1 promoter when KDM3A was depleted
by injecting 80 ng 3A MO. (F) Anti-Myc ChIPqPCR
analyses showing that ectopic Neurog2
was unable to bind the neurod1 promoter when
KDM3A was depleted (cMO or 3A MO: 80 ng).
ChIP-qPCR results shown in D-F were
combined from two biological repeats. Three
technical replicates were made in each
biological repeat. *P<0.05; **P<0.01;
***P<0.005, ns, not significant (two-tailed
Student’s t-test). (G) A summary of our current
findings that posit the roles for KDM3A in
regulating chromatin states and its
collaboration with Neurog2 to initiate neuronal
precursor differentiation. See details in the
Discussion.
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Fig. S3 (related to Fig. 2). KDM3A is required for primary neurogenesis in
Xenopus and depletion of KDM3A using a second MO (MO2). (A, B) Embryos at
stage 18 in situ hybridized with tubb2b (A, dorsal view) or neurod1 (B, dorsal anterior
view). 80 ng 3A MO or cMO was injected into both cells at the 2-cell stage. mRNA
encoding Xneurog1 (100 pg), Xneurog3 (100 pg), or mouse Neurog2 (50 pg) was
injected into two dorsal cells at the 4-cell stage. (C) A schematic depiction of KDM3A
MO and MO2 targeting different locations that are critical for the translation control
of kdm3a mRNA. (D) Western blot detection of KDM3A protein at stage 11 after
KDM3A MO2 injection at the 2-cell stage (40 and 80 ng). (E) Semi-quantitative PCR
analyses of gene expression in animal caps with indicated treatment. cMO: 80 ng. 3A
MO2: 80 ng. neurog2 mRNA: 100 pg. (F) Effects of KDM3A MO2 injection on
tubb2b expression analyzed through WISH (left column, dorsal view), neural tube
closure (middle column, dorsal anterior view, yellow arrows demarcate the closing
neural tubes), and later development (right column, lateral view, solid triangles
indicate eyes). cMO: 60 ng. 3A MO2: 60 ng. 3A MO2-resistant kdm3a mRNA: 500
pg. |
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Fig. S5 (related to Fig. 5). Assessment of the activities of Ascl1 on neuronal gene
expression and H3K9me2.
(A) Semi-quantitative PCR analyses of gene expression in animal cap explants treated
with Neurog2 or Ascl1. (B) Western blot detection of overexpressed 6MT-Neurog2
and 6MT-Ascl1 in the animal cap explants prepared through the same procedure as
done in (A). (C, C’) WISH (C) and RT-qPCR (C’) detection of gene expression in
control and Ascl1 splice blocking MO (Ascl1 sMO, 80 ng)-injected embryos at the
stage 18. * P<0.05; ** P<0.01; *** P<0.005, according to two-tailed Student t-test..
(D, E) Anti-H3K9me2 ChIP-qPCR analyses showing that 80 ng Ascl1 sMO did not
alter the H3K9me2 marks on the promoter regions of neurod1 (D) or tubb2b (E). (F)
ChIP-qPCR detection of KDM3A on the -36 bp position of tubb2b promoter.
6MT-neurog2 mRNA (500 pg) and 6MT-ascl1 mRNA (200 pg) were individually
injected at the 2-cell stage and embryos were then harvested at the stage 15 followed
by ChIP-qPCR procedures. (G-I) ChIP-qPCR data showing the effects of ectopic
Neurog2 and Ascl1 on the promoter region of myt1. Both ectopic Neurog2 and Ascl1
were able to bind myt1 promoter (G). Only overexpressed Neurog2 increased the level
of KDM3A (H), and decreased the H3K9me2 marks (I) on the promoter of myt1. *
P<0.05; ** P<0.01; *** P<0.005; ns: no significance, according to two-tailed
Student’s t-test. |
