Distal enhancer-insulator module of GDF6 is essential for cochlear formation
Mohammad Faraz Zafeer, Clemer Abad, Havva Ortabozkoyun, Memoona Ramzan, Guney Bademci, Maria C. Robayo, Duygu Duman, Rolen M. Quadros, Shengru Guo, Juan I. Young, Anthony J. Griswold, Channabasavaiah B. Gurumurthy, Derek M. Dykxhoorn, Katherina Walz, Mustafa Tekin
Mohammad Faraz Zafeer, Clemer Abad, Havva Ortabozkoyun, Memoona Ramzan, Guney Bademci, Maria C. Robayo, Duygu Duman, Rolen M. Quadros, Shengru Guo, Juan I. Young, Anthony J. Griswold, Channabasavaiah B. Gurumurthy, Derek M. Dykxhoorn, Katherina Walz, Mustafa Tekin
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Research Article Development Genetics

Distal enhancer-insulator module of GDF6 is essential for cochlear formation

Abstract

Several genes guide inner ear development, and mutations in these genes can cause malformations that result in congenital hearing loss. However, the contribution of noncoding regulatory elements remains largely unclear. This study investigates the function of distal enhancer elements in the transcriptional regulation of GDF6, a gene implicated in cochlear development. Using mouse models with targeted deletions, human inner ear organoids, and CRISPR interference (CRISPRi), we identified a downstream regulatory interval harboring a developmental enhancer required to maintain GDF6 expression during otic epithelial maturation and cochlear morphogenesis. Deletion of this regulatory region or targeting of CRISPRi-based repressors to these regions resulted in decreased GDF6 expression, failure of otic-epithelium development, and prevention of hair cell–like differentiation, reflecting cochlear aplasia observed in patients with corresponding genomic deletions. These findings highlight the contribution of long-range regulatory elements to auditory development and illustrate how their disruption contributes to human deafness.

Authors

Mohammad Faraz Zafeer, Clemer Abad, Havva Ortabozkoyun, Memoona Ramzan, Guney Bademci, Maria C. Robayo, Duygu Duman, Rolen M. Quadros, Shengru Guo, Juan I. Young, Anthony J. Griswold, Channabasavaiah B. Gurumurthy, Derek M. Dykxhoorn, Katherina Walz, Mustafa Tekin

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Figure 1

Characterization of Gdf6delFull–/– and Gdf6del3′–/– mice.

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Characterization of Gdf6delFull–/– and Gdf6del3′–/– mice. (A) Schematic ...
(A) Schematic of CRISPR/Cas9 sgRNA placement and the deleted regions in Gdf6delFull–/– (~221 kb; Chr4:10,249,919–10,471,132) and Gdf6del3′–/– (~106 kb; Chr4:10,364,802–10,471,054) mice, with the critical region (Chr4:10,249,919–10,364,802) indicated. Positions of the 221 kb forward, 106 kb forward, and common reverse genotyping primers are shown. (B) Whole-mount front and back views of the inner ear from WT, Gdf6del3′–/–, and Gdf6delFull–/– mice, showing preserved vestibular and cochlear structures in WT and Gdf6del3′–/–, and cochlear aplasia in Gdf6delFull–/– mice (n = 3 per genotype). The cochlear apex is indicated by arrows. (C) Auditory brainstem response (ABR) thresholds for click stimuli and tone bursts at 8, 16, and 24 kHz. Gdf6delFull–/– mice showed markedly elevated thresholds compared with WT mice across all frequencies (***P < 0.001 at CLICK, 8 kHz, and 16 kHz; **P < 0.01 at 24 kHz) and compared with Gdf6del3′–/– mice (**P < 0.01 at 8 kHz and 16 kHz; *P < 0.05 at 24 kHz; ns at CLICK), consistent with profound hearing impairment. Thresholds did not differ significantly between WT and Gdf6del3′–/– mice at any frequency (ns; n = 3 per genotype). (D) qRT-PCR analysis of Gdf6 mRNA (normalized to Gapdh) at E17 in inner ear, liver, lung, brain, and heart from Gdf6delFull–/– and WT embryos (n = 3 per genotype). A significant reduction in Gdf6 expression is restricted to the inner ear (*P < 0.05); all other tissue comparisons are nonsignificant (ns). Data are presented as mean ± SEM. Statistical comparisons were performed by 2-way ANOVA followed by Tukey’s post hoc test for multiple comparisons; only comparisons reaching statistical significance are annotated; all others are labeled ns.