Page 14 - 南京医科大学自然版

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神经定向分化过程中神经祖细胞相关程序增强，而 一步阐明上述神经分化阶段性失衡的分子机制，本
后续成熟相关程序受阻。 研究首先进行了BID⁃seq检测（图5A），并对d3阶段
2.5 Pus7 敲除通过 mRNA 假尿嘧啶化扰动神经连 的 BID⁃seq 与 RNA⁃seq 数据进行联合分析。考虑到
接相关基因程序 PUS7缺失可能导致部分靶转录本Ψ修饰水平下降，
假尿嘧啶化扰动神经连接相关基因程序为进 而Ψ修饰降低可能进一步影响 mRNA 稳定性、加工

A B
d 3 d 4 d 5
Transfer EBs
N2B27
N2B27+1/4 2i （⁃2i，⁃Lif） WT
d0 d2 d3 d5 250 μm

C Pus7⁃KO

100 500 μm
Time（days）
75 d0
50
（25.9%） 25 0 d3 D d0 Endoderm Mesoderm Ectoderm Pluripotency log2 FC
d5
PC2 -25 Group （KO/WT）
-50
WT
-75 KO d3 2
-100 0
d5
-100 -2
-100 -75 -50 -25 0 25 50 75 75 100 Foxa2 Sox17 Afp Cxcr4 Hhex Gata4 Bmp4 T Kdr Mitf Nodal Eomes Nes Lhx2 Sox10 Pax6 Sox1 Klf4 Sox2 Nanog Pou5f1 Myc
PC1（47.8%）
E
WT WT WT WT
Pus7⁃KO
4 Pus7⁃KO ** 1.5 *** 4 Pus7⁃KO * 1.5 Pus7⁃KO
Sox1 mRNA level 3 2 1 * * mRNA level 1.0 Sox17 mRNA level 3 2 1 mRNA level Klf4 0.5
1.0

0.5

0 Bmp4 0 0 0
d0 d3 d5 d0 d3 d5 d0 d3 d5 d0 d3 d5
WT WT WT WT
Pus7⁃KO Pus7⁃KO Pus7⁃KO Pus7⁃KO
10 2.0 3 *** 1.5
***
mRNA level 6 ** mRNA level 1.5 *** mRNA level 2 mRNA level 1.0
8
1.0
Pax6 4 2 Kdr 0.5 Gata4 1 * Sox2 0.5

0 0 0 0
d0 d3 d5 d0 d3 d5 d0 d3 d5 d0 d3 d5
A：Schematic illustration of the experimental workflow for spontaneous EB differentiation of mESCs. B：Representative bright⁃field images of WT
and Pus7⁃KO mESC⁃derived EB at the indicated time points during differentiation. Magnified views of the indicated regions at day 4 are shown in the in⁃
sets. Scale bar=500 μm for overview images and 250 μm for magnified images. C：Principal component analysis（PCA）of RNA⁃seq samples from WT
and Pus7⁃KO cells during EB differentiation（n=2）. D：Heatmap showing the log 2FC of pluripotency⁃ and germ layer⁃associated marker genes in Pus7⁃
KO relative to WT，based on RNA⁃seq analysis at d0，d3，and d5 during EB differentiation（n=2）. E：qRT⁃PCR analysis of the mRNA expression of ecto⁃
derm/neuroectoderm marker genes（Sox1 and Pax6），mesoderm marker genes（Bmp4 and Kdr），endoderm marker genes（Sox17 and Gata4），and pluripo⁃
**
*
tency marker genes（Klf4 and Sox2）in WT and Pus7⁃KO mESC at the indicated time points during EB differentiation（n=3）. P < 0.05，P < 0.01，and
***
P < 0.001.
图3 Pus7敲除促进EB分化过程中的外胚层谱系倾向
Figure 3 Pus7 knockout biased EB differentiation toward the ectodermal lineage