Fig 1: Crtl1 promoter activity is regulated by Mef2c.Fold change in luciferase activity driven by approximately 1 kb of the Crtl1 promoter in the pGL3 luciferase reporter vector was assayed in fetal chicken VICs (A and B) and in NIH3T3 cells (C and D). In fetal chicken VICs (A) and NIH3T3 cells (C), Crtl1 promoter activity was significantly increased with increasing concentrations of Mef2c. (B) Crtl1 promoter activity in the presence of 100 ng Mef2c with the addition of 200 ng of the Mef2-Engrailed dominant negative expression construct resulted in an approximately 30% reduction in Crtl1 reporter activity. (D) Mutations were introduced into the Crtl1 promoter construct at Mef2 Site 1 and Mef2 Site 2 (Crtl1-Mutant 1 and Crtl1-Mutant 2 respectively). Crtl1-Mutant 1 results in an approximately 30% reduction in Crtl1 promoter activation in the presence of 100 ng of Mef2c and Crtl1-Mutant 2 results in an approximately 50% reduction of Crtl1 activity.(*p<0.05, #p<0.1).
Fig 2: MEF2C is a target of miR-21. (a) Alignment and base pairing of miR-21 with the MEF2C 3'-UTR. The seed region (bases 2–8) is indicated in capital letters. In vitro mutagenesis of the wild-type (wt) MEF2C sequence (upper) to alter the seed sequence and create the mutant (mut) sequence in the seed region is shown in italics (lower). (b) MiR-21 significantly lowers the activity of the wild-type MEF2C luciferase reporter, but not the mutant MEF2C or a random DNA sequence. This experiment was repeated in three independent experiments. (c) HEK293T cells or (d) human embryonic neurons were transduced with either control FUGW or miR-21-FUGW lentiviral vector. Cells were immunostained for MEF2C, as seen in the second panel, the reactivity for MEF2C decreases in miR-21 transduced cells (630 × magnification, bar=10 µm). Mean and standard error of the mean shown, significance is indicated by **P<0.01
Fig 3: Enhanced NOTCH activation and efficient radial organization co-localize with cortical markers in organoids derived by Triple-i.a, Merged bright-field images and their matched H9 HES5::eGFP confocal images taken of representative day 17 organoids (n = 9). Maximum intensity projections for H9 HES5::eGFP confocal images taken at intervals of 15 µm are shown. Green arrows, enhanced NOTCH-active and radially organized regions; white arrows, radially organized regions lacking enhanced NOTCH activation; red arrows, non-neuro-epithelial extensions. b, Mean HES5::eGFP intensity levels measured for the images from a (n = 9). c, Number of rosettes expressing HES5::eGFP in the organoid images from a. b,c, Statistical test, two-sided t-test with Benjamini–Hochberg correction; *P < 0.05; ***P < 0.001; ****P < 0.0001; and NS, not significant. The boxplots display the median and interquartile range (box boundaries) with whiskers extending to 1.5× the interquartile range; n = 9. d, Immunostaining of representative H9-derived day 30 organoids (left) and counts within rosettes of SOX1, FOXG1 and HES5::eGFP (top right; Triple-i, n = 13 from two replicates; Dual SMAD-i, n = 4 from one replicate; and Inhibitor-free, n = 5 from one replicate) as well as PAX6, EMX2 and HES5::eGFP (bottom right; Triple-i and Dual SMAD-i, n = 10 from one replicate; and Inhibitor-free, n = 18 from two replicates). e, Immunostaining of representative ZIP8K8-derived day 27 organoids (left) as well as counts within rosettes of EMX2 (Triple-i, n = 8 from one replicate; Dual SMAD-i, n = 38 from three replicates; and Inhibitor-free, n = 14 from one replicate), SP8 (Triple-i, n = 35 from three replicates; Dual SMAD-i, n = 23 from three replicates; and Inhibitor-free, n = 12 rosettes from one replicate) and NR2F1 (Triple-i, n = 36 from three replicates; Dual SMAD-i, n = 44 from three replicates; and Inhibitor-free, n = 12 from one replicate) with PAX6, FOXG1 and SOX1 (Triple-i, n = 40 from two replicates; Dual SMAD-i, n = 29 from two replicates; and Inhibitor-free, n = 14 from one replicate; right). f, Immunostaining of representative ZIP8K8-derived day 27 organoids (left) and counts within rosettes of the cortical marker MEF2C with SOX2 and DCX (top right; Triple-i, n = 19 from two replicates; Dual SMAD-i, n = 22 from two replicates; and Inhibitor-free, n = 9 from one replicate), OLIG3 and TCF7L2 (middle right; Triple-i and Dual SMAD-i, n = 7 from one replicate; and Inhibitor-free, n = 11 from one replicate), and TTR and LMX1A (bottom right; Triple-i, n = 10; Dual SMAD-i, n = 13; and Inhibitor-free, n = 9; all from one replicate). a,d–f, Scale bars, 100 µm. d–f, The bars represent the mean. Immunostaining counts for b–f are provided.Source data
Fig 4: Photomicrographs of monkey and human brain tissue. Immunohistochemical staining for MEF2C reveals positive staining in hippocampal (a) and cortical (b) neurons of control uninfected monkeys. In monkeys with SIVE much less reactivity is seen in hippocampal (c) and cortical neurons (d). Tissue from four monkeys in each group was examined. The same result is seen in the cortex of humans, with stronger reactivity is seen in neurons in the frontal cortex of brains from individuals without HIV neurological symptoms (e and f, representing two of the four cases examined) when compared with those with HIV-associated dementia (g and h, representing two of the three cases examined). The chromagen revealing the positive reaction is brown, and methyl green (light green) used for counterstaining. Original magnification 200 × (bar=20 µm) except inserts at 630 × (bar=10 µm)
Fig 5: Sequence alignment of the mouse, rat, and human Crtl1 (Hapln1) promoters.The mouse, rat, and human Crtl1 genes, plus 1000 bp of the upstream promoter, were aligned using the web-based tool Kalign. Using this alignment, two conserved Mef2 consensus sites were identified at positions -698 to -707 and -913 to -923.
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