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POU3F4 is a member of the POU-homedomain transcription factor family with a prominent role in inner ear development. Mutations in the human POU3F4 coding unit leads to X-linked deafness type 3 (DFN3), characterized by conductive hearing loss and progressive sensorineural deafness. Microdeletions found 1 Mb 5' upstream of the coding region also displayed the same phenotype, suggesting that cis-regulatory elements might be present in that region. Indeed, we and others have recently identified several enhancers at the 1 Mb 5' upstream interval of the pou3f4 locus. Here we characterize the spatio-temporal patterns of these regulatory elements in zebrafish transgenic lines. We show that the most distal enhancer (HCNR 81675) is activated earlier and drives GFP reporter expression initially to a broad ear domain to progressively restrict to the sensory patches. The proximal enhancer (HCNR 82478) is switched later during development and promotes expression, among in other tissues, in sensory patches from its onset. The third enhancer (HCNR 81728) is also active at later stages in the otic mesenchyme and in the otic epithelium. We also characterize the signaling pathways regulating these enhancers. While HCNR 81675 is regulated by very early signals of retinoic acid, HCNR 82478 is regulated by Fgf activity at a later stage and the HCNR 81728 enhancer is under the control of Hh signaling. Finally, we show that Sox2 and Pax2 transcription factors are bound to HCNR 81675 genomic region during otic development and specific mutations to these transcription factor binding sites abrogates HCNR 81675 enhancer activity. Altogether, our results suggest that pou3f4 expression in inner ear might be under the control of distinct regulatory elements that fine-tune the spatio-temporal activity of this gene and provides novel data on the signaling mechanisms controlling pou3f4 function.
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21209840
???displayArticle.pmcLink???PMC3013142 ???displayArticle.link???PLoS One
Figure 1. Temporal expression pattern of GFP driven by pou3f4 HCNR 81675 and HCRN 82478 enhancers.(A) Schematic representation of the POU3F4 locus in the human chromosome X (hg18 alignment) showing the position of the different inner ear enhancers relative to the POU3F4 coding sequence. (BâC) Onset of GFP protein expression in HCNR 81675 (B) and HCNR 82478 (C) transgenic embryos. Expression in the otic territory occurs at 13.5 hpf in HCNR 81675 and at 18.5 hpf in HCNR 82478 zebrafish embryos (white arrows), GFP in mesonephros and midbrain-hindbrain boundary (red arrows) is also detected in HCNR 82478 embryos. (DâE) Dorsal views of HCNR 81675 (D) and HCNR 82478 (E) transgenic embryos assayed by in-situ hybridization for GFP mRNA expression. In both cases GFP mRNA was detected in the otic field 2 hours before GFP protein was found (BâC). Orientation of the embryos is anterior (left) to posterior (right).
Figure 2. Spatial-temporal expression pattern of pou3f4 enhancers in the inner ear.(AâF) Lateral views of inner ears from zebrafish transgenic embryos for HCNR 81675 (AâC) and HCNR 82478 (DâF) enhancers analysed from 24 hpf to 72 hpf. In HCNR 81675 embryos at 24 hpf, GFP is observed in two broad domains comprising the sensory territories as observed by the otolith deposition (stars) (B). In HCNR 82478, GFP is already restricted to the anterior and posterior sensory macula from its onset as observed by GFP fluorescence relative to the otolith position (star). (C and F) GFP is found in the three sensory crista in 3-day old embryos in both transgenic zebrafish lines. Orientation is anterior (left) and dorsal (up). (G and H) Confocal transverse images of inner ear sensory patches immunostained with the anti-Pax2 antibody in 72 hpf embryos. In HCNR 81675 embryos, GFP is found in supporting cells but absent in hair-cells (Pax2 positive cells; pointed by a white arrow) (G). In contrast, HCNR 82478 embryos displayed GFP in supporting cells but also in hair-cells at lower levels (white arrow) whereas other hair-cells where completely devoid of GFP expression (red arrow). (I and J) Transverse confocal images of sensory patches of both enhancer embryos immunostained for GFP after the injection of the hair cell specific labelling marker FM 4-64FX. The same result was obtained in this experiment. (I) GFP is devoid in FM 4-64FX stained hair-cells in HCNR 81675 embryos (white arrow), whereas some hair-cells displayed GFP in HCNR 82478 embryos (J; white arrow). (K and L) Confocal images taken from the transverse section anterior to the first section from the otic vesicle. Co-immunostaining for anti-GFP and anti-islet1 protein reveals that only in HCNR 82478 transgenic embryos GFP is activated in the otic ganglion (L). (GâL) Lateral (left) and dorsal (up).
Figure 3. Distinct signaling pathways regulate activation of pou3f4 HCNR 81675 and HCNR 82478 enhancers.(AâN) Transgenic embryos for both enhancers were treated with different pharmacological inhibitors from 5.5 hpf stage to 18â20 hpf and 7.5 hpf to 36â40 hpf respectively. Lateral view of HCNR 81675 (AâG) 18â20 hpf staged otic vesicles and HCNR 82478 (FâN) otic vesicles of 36â40 hpf embryos. HCNR 81675 activity was abrogated in the presence of RA signaling inhibitor DEAB (compare D to the control treatment with DMSO in A), whereas Fgf signaling inhibition by SU5402 completely disrupted pou3f4 HCNR 82478 activity (compare I to control treatment in H). Orientation is anterior (left) to posterior (right). (O, P) Graphs representing the percentage of embryos displaying complete inhibition of GFP expression in pou3f4 HCNR 81675 (O) and HCNR 82478 (P) transgenic embryos after specific signaling pathway blockade. The total number of embryos counted in three independent experiments is represented.
Figure 4. The POU3F4 HCNR 81728 enhancer is regulated by Hedgehog signaling.(A and B) Lateral view of GFP otic expression in 96 hpf embryos transgenic for the HCNR 81728 enhancer in control (A) and Cyclopamine A treated embryos (B). (C) Percentage of GFP expressing area in otic vesicles from 95% EtOH and Cyclopamine A treated embryos.
Figure 5. Co-localization of Pax2 and Sox2 with GFP driven by the HCNR 81675 enhancer.(AâD) Dorsal view of transgenic embryos assayed by ISH for the expression of pax2a (A), sox2 (B), sox10 (C) at 13, 15 and 18 hpf. GFP (D) displays a similar pattern than sox2 and pax2a at 15 hpf (compare A and B with D). Orientation is anterior to the left. (EâEâ³) Double immunostaining with anti-Pax2 (E) and anti-GFP antibody (Eâ²) in transverse sections of 15 hpf otic vesicles revealed co-localization of both proteins (Eâ³). (FâFâ³) GFP protein (Fâ²) also co-localizes with sox2 mRNA (Fâ³) but not sox10 or tbx1 mRNA (Gâ³ and Hâ³). (IâP) pax2a and sox2 expression is abolished in retinoic acid treated HCNR 81675 embryos (compare J and L to I and K, respectively) but not other genes such as sox10 or neuroD (compare N and P to M and O, respectively). Dorsal view, orientation is anterior to the left.
Figure 6. Pax2 and Sox2 are directly recruited to the HCNR 81675 DNA.(A) rVista 2.0 alignment of HCNR 81675 human and Xenopus genomic sequence. Conserved binding sites for Sox and Pax2/5/8 proteins found by TRANSFAC are represented. Primer location enclosing the genomic region of Sox and Pax2/5/8 binding sites designed for chromatin immunoprecipitation are marked with red and violet arrows respectively. (B) ChIP with anti-Pax2 and anti-Sox2 antibodies from stage 30â34 Xenopus otic vesicles was performed and the PCR amplification of the DNA fragments pulled down by Pax2 and Sox2 chromatin immunoprecipitation is shown. A region of the haemoglobin locus with no Pax2 and Sox2 binding sites shows no immunoprecipitation with these antibodies. (C) Graphs representing the relative fold enrichment of Sox2 and Pax2 binding to the HCNR 81675 but not to the haemoglobin region detected by quantitative PCR.
Figure 7. Pax2 and Sox2 proteins are required for HCNR 81675 enhancer activation.(A) Scheme showing wild-type Sox and Pax2/5/8 consensus in the pou3f4 HCNR 81675 sequence and above each one, the mutation in the primers designed for site-directed mutagenesis of the Sox and Pax2/5/8 binding sites. (BâC) Transgenic embryos carrying GFP under the control of the HCNR 81675 enhancer. (B) GFP expression promoted by the wild type HCNR 81675 sequence. (C) GFP expression promoted by the HCNR 81675 enhancer harbouring the double mutation for Pax2/5/8 and Sox binding sites.
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