(
A) Pou5f3 morpholino validation. Brightfield images of embryos that were injected with control or Pou5f3 morpholino. Embryos injected with Pou5f3 morpholino arrested at sphere or dome stage, as reported previously (
Burgess et al., 2002). Scale bar, 250 μm. Western blot of embryos injected with 50 ng
pou5f3-2xHA mRNA alone or in combination with 6 ng Pou5f3 moprholino. As expected, the morpholino reduces the expression of Pou5f3. (
B) We injected embryos with Pou5f3 morpholino and analyzed the effect on transcription. Shown are the fold changes in relative expression levels of Pou5f3-morpholino-injected embryos compared to control morpholino-injected embryos at high stage for Pou5f3 targets
apoeb,
dusp6,
klf17,
irx7, and
klf2b, and non-Pou5f3 targets
sox19a,
grhl3 and
gadd45bb. Reduction of Pou5f3 results in decreased transcription for all Pou5f3 target genes analyzed at high stage, confirming that they are regulated by Pou5f3. Non-targets were not affected. Error bars represent SEM (n = 4). n.s. p>0.05; *p<0.05; **p<0.01; ***p<0.001 (two-tailed Student’s t-test, compared to control morpholino). (
C) Expression of
klf17,
irx7 and
klf2b was analyzed by qPCR at 512-cell, early 1K, mid 1K and high stage in control and Pou5f3 morpholino-injected embryos. Bar graphs focus on mid 1K stage. Error bars represent SEM (n ≥ 4). **p<0.01; **p<0.001 (two-tailed Student’s t-test, compared to control morpholino). (
D) Western blot analysis of embryos injected with
sox19b-2xHA mRNA alone and in combination with 2 ng of Sox19b morpholino validate the effect of the Sox19b morpholino. (
E) Expression of
dusp6 was analyzed by qPCR at 512-cell, early 1K, mid 1K and high stage in control and Sox19b morpholino-injected embryos.
dusp6 was selected for analysis because it is the only gene from our selected Pou5f3-target genes that is also regulated by SoxB1 (
Lee et al., 2013). Error bars represent SEM (n ≥ 4). (
F) Validation of FoxH1 target genes. To verify that the genes we selected require FoxH1 for their expression, we injected embryos with FoxH1 morpholino and analyzed the effect on transcription. Shown are the fold changes in relative expression levels of FoxH1 morpholino-injected embryos compared to control morpholino-injected embryos at high stage for FoxH1 targets
dusp6,
flh,
wnt11 and
gadd45bb. Reduction of FoxH1 results in decreased transcription for all FoxH1 target genes. (
G) Expression of
dusp6, flh, wnt11 and
gadd45bb was analyzed by qPCR at 512-cell, early 1K, mid 1K and high stage in control and FoxH1 morpholino-injected embryos. All four target genes show a delay in the time when they are first transcribed in the FoxH1 morphants compared to control morpholino-injected embryos. Error bars represent SEM (n ≥ 4). (
H) Staging by morphology was verified by cell counting at the stages used for the Pou5f3 morpholino analysis. Each data point represents a single embryo. Error bars represent SEM. Gene expression is normalized to the expression of
eif4g2α.