Additional file 1 of Zmynd11 is essential for neurogenesis by coordinating H3K36me3 modification of Epha2 and PI3K signaling pathway
Creators: name: Yang, Xu
name: Li, Lan
name: Qu, Wenzheng
name: Cheng, Xuejun
name: Zhang, Jinyu
name: Sun, Yan
name: Liu, Suxiao
name: Peng, Guoping
name: Zheng, Rui
name: Li, Xuekun
Publisher: name: figshare
Year: 2025
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Developmental Biology
subject: Mental Health

description: Supplementary material 1: Figure S1. Dynamic and abundant expression of Zmynd11 in embryonic brain and neuronal progenitor cells.Representative images of Zmynd11 and embryonic neuronal progenitor cellsmarker Nestin, neuronal cell marker Tuj1 immunostaining with brain sections of embryonic miceand postnatal day 0 mice. Scale bars, 50 μm. SVZ, subventricular zone; VZ, ventricular zone; IZ, intermediate zone. Representative images of WBand quantification resultsshowed that the level of Zmynd11 was significantly increased in the cortex with neuronal development of mice. GAPDH was used as an internal control. n = 5 mice in each group. Data are presented as mean ± SEM; *p &lt; 0.05; **p &lt; 0.01; ***p &lt; 0.001, ****p &lt; 0.0001, one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F= 62.19. qRT-PCR assay results showed the level of Zmynd11 was significantly increased in the cortex of mice with neuronal development. n = 3 biologically independent experiments. Data are presented as mean ± SEM; *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F= 116.3. Representative images of Zmynd11, Nestin and Tuj1 immunostaining with proliferating and differentiated eNPCs, respectively. Scale bars, 50 μm. Representative images of WB and quantification results showed that the level of Zmynd11 was significantly increased upon the differentiation of eNPCs. GAPDH was used as an internal control. n = 5 mice in each group. Data are presented as mean ± SEM; *p &lt; 0.05; **p &lt; 0.01; ***p &lt; 0.001, ****p &lt; 0.0001, unpaired Student’s t test.qRT-PCR assay results showed the level of Zmynd11 was significantly increased upon the differentiation of eNPCs. n = 3 biologically independent experiments. Data are presented as mean ± SEM; *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, unpaired Student’s t-test.Representative images of Zmynd11, Nestin, neuronal cell marker MAP2 and astrocyte cell marker GFAP immunostaining with proliferating eNPCs, neurons and astrocytes respectively. Scale bars, 50 μm.Representative images of WBand quantification resultsshowed that the level of Zmynd11 in neurons and astrocytes was significantly increased compared to proliferating eNPCs. GAPDH was used as an internal control. n = 5 mice in each group. Data are presented as mean ± SEM; *p &lt; 0.05; **p &lt; 0.01; ***p &lt; 0.001, ****p &lt; 0.0001, one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F= 61.14.qRT-PCR assay results showed the level of Zmynd11 in neurons and astrocytes was significantly increased compared to proliferating eNPCs. n = 3 biologically independent experiments. Data are presented as mean ± SEM; *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F= 106.2. Figure S2. Specific deficiency of Zmynd11 in Nestin+ cells results in developmental deficits of mice. Schematic diagram of the generation of Zmynd11 cKO mice.Representative images of Ctrl and cKO mice at the age of P0. Quantification of body weight of Ctrl and cKO mice at P0. n = 6 mice for each group. Data are presented as mean ± SEM; *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, unpaired Student’s t-test. Representative images of radial glial marker PAX6 immunostaining with scramble and Zmynd11 KD brain sections. Scramble and shZmynd11 plasmids were delivered into the brain via in utero electroporation at E14, and mice were sacrificed for assay at E17. Scale bar, 50 μm. Quantification results showed the percentage of PAX6+GFP+/GFP+ in VZ/SVZ layer was significantly decreased in Zmynd11 KD group compared to Ctrl. n = 5 fetuses at least from 4 pregnant mice for each group. Data are presented as mean ± SEM; *p &lt; 0.05; **p &lt; 0.01, unpaired Student’s t test.Representative images of intermediate progenitor marker Tbr2 immunostaining with scramble and Zmynd11 KD brain sections. Scale bar, 50 μm. Quantification results showed the percentage of Tbr2+GFP+/GFP+ in VZ/SVZ layer was significantly increased in Zmynd11 KD group compared to Ctrl. n = 5 fetuses at least from 4 pregnant mice for each group. Data are presented as mean ± SEM; *p &lt; 0.05; **p &lt; 0.01, ***p &lt; 0.001, unpaired Student’s t test. Representative images of the distribution of GFP+ cells in the cortex of E17 fetal brain. Scramble and shZmynd11 plasmids were delivered into E14 mice brains via IUE, and mice were sacrificed for assays at E17. Scale bar, 50 μm. Quantification results showed that Zmynd11 KD induced fewer GFP+ cells in VZ/SVZ and CP regions, but more cells in IZ compared to scramble group. n = 5 fetuses at least from 4 pregnant mice for each group. Data are presented as mean ± SEM; *p &lt; 0.05; **p &lt; 0.01, ***p &lt; 0.001, unpaired Student’s t test. Figure S3. Zmynd11 KD inhibits PI3K signaling pathway in eNPCs. Schematic diagram of genes relating to PI3K signaling pathway.qRT-PCR assay results showed that Zmynd11 KD significantly reduced the mRNA level of multiple key components of PI3K signaling pathway in eNPCs. n = 3 biologically independent experiments. Data are presented as mean ± SEM; *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, n.s. no significance; unpaired Student’s t-test. Representative images of WB of PI3K components in proliferating eNPCs infected with lenti-scramble and lenti-shZmynd11, respectively. Cells were collected for assays 3 days post the virus infection. Quantification results showed that Zmynd11 KD significantly reduced the level of Epha2. Although the levels of total IRS1, PI3K, PDPK1, and SGK1were not changed, the levels of Epha2, p-IRS1, p-PI3K, p-PDPK1, and p-SGK1were significantly decreased in eNPCs. n = 4 biologically independent experiments. Data are presented as mean ± SEM; *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, unpaired Student’s t-test. Figure S4. Zmynd11 deficiency did not affect total levels of H3K36me3 and Pol II. Immunoprecipitation followed by WB assays showed that Zmynd11 easily precipitated H3K36me3 in eNPCs, and vice versa. ChIP followed by WB assays showed the enrichment of H3K36me3 at Epha2 promotor in eNPCs.WB assays and quantification results showed that the level of total H3K36me3 was not changed in cKO brain tissues and Zmynd11 KD eNPCs. H3 was used as internal control. n = 4 biologically independent experiments. Data are presented as mean ± SEM; *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, unpaired Student’s t-test.ChIP followed by WB assay showed the binding of RNA Polymerase II at Epha2 promotor in eNPCs. WB assay and quantification results showed that levels of Pol II was not changed in cKO brain tissues. Tubulin was used as internal control. n = 4 biologically independent experiments. Data are presented as mean ± SEM; *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, unpaired Student’s t-test. Figure S5. Exogenous Epha2 rescues neuronal deficits induced by Zmynd11 KD. Cortical neurons from E14 fetal mice were infected with lentivirus at DIV 4, and MAP2 immunostaining was performed at DIV 7. Lenti-scramble-GFP, Lenti-RFP, Lenti-shZmynd11-GFP, and lenti-Epha2-RFP viruses were used for infecting cells. Scale bar, 50 μm. Quantification results showed that exogenous Epha2 significantly enhanced the dendritic lengthand the number of branch pointsof cortical neurons. n = 30 cells for each group. Data are presented as mean ± SEM; *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F= 44.25 for, F= 42.61 for. Sholl analysis results showed that exogenous Epha2 significantly increased the dendritic complexity of cortical neurons. n = 30 cells for each group. Data are presented as mean ± SEM; *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F= 10.53. qRT-PCR assay results showed that exogenous Epha2 did not affect the level of Zmynd11 mRNA, but significantly enhanced mRNA levels of Epha2 and Gfap, and reduced the mRNA level of Tuj1. n = 3 biologically independent experiments. Data are presented as mean ± SEM; *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F= 82.44 for, F= 5374 for, F= 71.21 for, F= 107.8 for.