Fig 1: Nox2 (NADPH oxidase-2)-dependent upregulation of GDF6 (growth differentiation factor 6). A, GDF6 levels in conditioned medium of primary cardiac fibroblasts infected with adenoviruses expressing shRNA (short hairpin RNA) targeted against Nox2 (Ad.shNox2) or adenoviruses expressing shRNA targeted against a GFP (green fluorescent protein) control (Ad.shGFP; control) and treated with ANG II (angiotensin II) or vehicle. Representative immunoblot shown at the top and mean data at the bottom; n=3 per group. Ponceau staining of the membrane is shown in Figure XI in the Data Supplement. B, GDF6 protein levels in aorta from Fibro-Nox2KO or matched control (Nox2Flox) mice chronically treated with ANG II or saline vehicle. Representative immunoblot shown at the top and mean data at the bottom; n=4 per group. β-actin was used as a loading control. Densitometry values for GDF6-specific bands were divided by the corresponding densitometry of Ponceau bands in A and β-actin bands in B. Arbitrary units were calculated by normalizing data vs the control group (ie, fold-change). C, GDF6 immunostaining in aorta from Fibro-Nox2KO and control mice treated with ANG II or saline. Representative images are shown on the left and mean fluorescence intensity on the right. GDF6, red; 4′-6-diamidino-2-phenylindole (DAPI), blue; autofluorescence (AF), green. Scale bars=50 µm. 1-way ANOVA followed by Tukey post-test. L indicates vessel lumen. *P<0.05.
Fig 2: GDF6 (growth differentiation factor 6) induces vascular smooth muscle cell (VSMC) growth. A, Concentration-dependent effect of recombinant GDF6 (0–4 μg/mL) on BrdU (bromodeoxyuridine) incorporation in VSMC; n=4–6 per group. B, Representative Western blot for GDF6 expression after silencing using siRNA (silencing RNA) against GDF6 or scrambled control (control) in primary cardiac fibroblasts. C and D, VSMC growth assessed by Ki67 staining after incubation with conditioned media of aortic (C) or cardiac (D) fibroblasts treated with ANG II (angiotensin II). Some fibroblasts were treated with siGDF6 (silencing RNA against GDF6) to reduce GDF6 levels. 1-way ANOVA followed by Tukey post-test (A and C) or Kruskal-Wallis followed by Dunn post-test (D). *P<0.05.
Fig 3: GDF6 prevents PE-induced hypertrophic growth of NRVMs. (A) Immunofluorescence staining of α-actinin and quantification of cell area in NRVMs with or without GDF6 knockdown. (B) Nppa, Nppb, Myh7, and Myh6 mRNA levels in NRVMs with or without GDF6 knockdown. (C) GDF6 mRNA levels in NRVMs with or without GDF6 knockdown. (D) Immunofluorescence staining of α-actinin and quantification of cell area in NRVMs with or without recombinant human GDF6 (rhGDF6) treatment. (E) Nppa, Nppb, Myh7, and Myh6 mRNA levels in NRVMs with or without rhGDF6 treatment. n = 6 per group. * p < 0.05 versus matched PBS NRVMs receiving siCtrl or dimethyl sulfoxide (DMSO) treatment, # p < 0.05 versus matched PE NRVMs receiving siCtrl or DMSO treatment. One-way ANOVA followed by Tukey post hoc test was applied. In (C), Student’s two-tailed t-test was performed.
Fig 4: CRISPRi perturbation of a GDF6 regulatory region impairs otic epithelial organization and hair cell differentiation in IEOs.(A) Representative confocal immunofluorescence images (n = 3; 20× magnification; scale bar: 50 µm) of day 8 IEOs derived from parental CD2dCas9, CD2dCas9-CTCF (targeting the putative CTCF site), and CD2dCas9-CAN (targeting the candidate enhancer) lines, stained for DAPI (blue), SOX2 (orange), TFAP2A/AP2 (magenta), and CDH1/ECAD. (B) qRT-PCR analysis of GDF6 mRNA expression in IEOs at days 8, 12, and 15, normalized to HPRT1. Both CD2dCas9-CTCF and CD2dCas9-CAN lines show significantly reduced GDF6 expression compared with parental CD2dCas9 at all 3 time points (day 8: ***P < 0.001 for CTCF, **P < 0.01 for CAN; day 12: ***P < 0.001 for CTCF, *P < 0.05 for CAN; day 15: *P < 0.05 for CTCF, ***P < 0.001 for CAN). Data are presented as mean ± SEM from 3 biological replicates (each pooled from ~8 organoids) per sample. Statistical comparisons were performed by 1-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. (C) Representative confocal immunocytochemistry of day 55 IEOs from CD2dCas9, CD2dCas9-CTCF, and CD2dCas9-CAN lines stained for DAPI (blue), SOX2, MYO7A (magenta), and ESPN. The top row shows whole-organoid overviews (n = 2; 20× magnification); the bottom row shows high-magnification views (n = 2; 40× magnification; scale bar: 50 µm) of the same samples.
Fig 5: Chromatin accessibility and epigenomic profiling reveal CTCF binding sites and enhancer loss downstream of GDF6 in cochlear aplasia.(A and B) ATAC-Seq profiles in control-derived inner ear organoids at day 8 (A) and day 13 (B) showcase dynamic open chromatin regions downstream of the GDF6 locus (chr8: ~95.72–95.75 Mb, hg38), highlighted with dashed ovals. ATAC-Seq data are from 1 representative biological replicate at day 8 and day 13. The tracks are shown to illustrate relative accessibility patterns at 2 time points as indicated. The orange arrow indicates the previously identified region in scATAC-Seq in 2D otic progenitor model as shown in Supplemental Figure 7. Black bars under the peaks indicate the statistically significant peaks called in ATAC-Seq. (C) Normalized ChIP-Seq densities (CTCF, H3K27ac, and H3K4me1) across approximately 250 kb downstream of GDF6 (chr8: 95,214,667–96,196,827) in control (CD-2) and patient (166-101) day 8 IEOs. The control shows strong enhancer signatures and CTCF binding at the candidate locus, which are absent in patient samples. The patient-derived deleted genomic region containing CTCF binding sites and CFAP418-AS1 lncRNA is indicated with a red line; the GDF6 locus is shown with dashed lines. The region shown was selected based on the pathogenic human deletions downstream of GDF6 and is not derived from genome-wide peak selection. The absence of enhancer-associated histone marks and CTCF signal in patient tracks compared with WT reflects deletion of the underlying genomic region shown with a red line. (D and E) Normalized ChIP-Seq densities for CTCF, H3K27ac, and H3K4me1, as well as ATAC-Seq densities around CTCF binding sites/candidate enhancers in the patient-derived deleted genomic region (D) and at the GDF6 locus (E) in patient IEOs compared with control at day 8. ATAC-Seq peaks overlapping with enhancer-associated marks and CTCF binding are shown with colored dashed lines in green and orange, respectively. CTCF binding sites are illustrated with red arrows. ChIP-Seq data are from 1 representative of 2 biological replicates for the control (CD-2) and 1 biological replicate for patient 166-101.
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