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. 2012 Jun;139(11):1965-77.
doi: 10.1242/dev.071670. Epub 2012 Apr 18.

EYA1 and SIX1 drive the neuronal developmental program in cooperation with the SWI/SNF chromatin-remodeling complex and SOX2 in the mammalian inner ear

Mohi Ahmed  1 , Jinshu Xu, Pin-Xian Xu
Affiliations

EYA1 and SIX1 drive the neuronal developmental program in cooperation with the SWI/SNF chromatin-remodeling complex and SOX2 in the mammalian inner ear

Mohi Ahmed et al. Development. 2012 Jun.

Abstract

Inner ear neurogenesis depends upon the function of the proneural basic helix-loop-helix (bHLH) transcription factors NEUROG1 and NEUROD1. However, the transcriptional regulation of these factors is unknown. Here, using loss- and gain-of-function models, we show that EYA1 and SIX1 are crucial otic neuronal determination factors upstream of NEUROG1 and NEUROD1. Overexpression of both Eya1 and Six1 is sufficient to convert non-neuronal epithelial cells within the otocyst and cochlea as well as the 3T3 fibroblast cells into neurons. Strikingly, all the ectopic neurons express not only Neurog1 and Neurod1 but also mature neuronal markers such as neurofilament, indicating that Eya1 and Six1 function upstream of, and in the same pathway as, Neurog1 and Neurod1 to not only induce neuronal fate but also regulate their differentiation. We demonstrate that EYA1 and SIX1 interact directly with the SWI/SNF chromatin-remodeling subunits BRG1 and BAF170 to drive neurogenesis cooperatively in 3T3 cells and cochlear nonsensory epithelial cells, and that SOX2 cooperates with these factors to mediate neuronal differentiation. Importantly, we show that the ATPase BRG1 activity is required for not only EYA1- and SIX1-induced ectopic neurogenesis but also normal neurogenesis in the otocyst. These findings indicate that EYA1 and SIX1 are key transcription factors in initiating the neuronal developmental program, probably by recruiting and interacting with the SWI/SNF chromatin-remodeling complex to specifically mediate Neurog1 and Neurod1 transcription.

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Figures

Fig. 1.
Fig. 1.
Otic neurogenesis is blocked in mice lacking Eya1 and Six1. (A-D) Whole-mount ISH for Neurog1 in wild-type (A), Eya1–/– (B), Six1–/– (C) and Eya1–/–;Six1–/– (D) embryos at E9.25. (E-L) Whole-mount ISH for Neurod1 in wild-type (E,I), Eya1+/–;Six1–/– (F), Six1–/– (G), Eya1–/– (J) and Eya1–/–;Six1–/– (H,K,L) embryos at E9.25-9.5. (M-P) Whole-mount ISH for Dlx5 in wild-type control (M) and Eya1;Six1 mutant (O) at E9.5. N and P show sections through otocyst regions of the embryos shown in M and O, respectively. Arrows point to the reduced expression in the otocyst (ov) or VIIIth ganglion. V, trigeminal (Vth) ganglion. Scale bars: 100 μm.
Fig. 2.
Fig. 2.
Eya1 and Six1 are expressed in otic neuroblast precursors and spiral ganglion. (A-F) Sections of X-gal-stained E8.75 Eya1lacZ/+ embryo (A), E14.5 Eya1lacZ/+ cochlear (B), E16.5 Eya1lacZ/+ cochlear stained with anti-NEUROD1 (red, C), E9.5 Six1lacZ/+ embryo (arrow points to differentiating neurons within the VIIIth ganglionic anlagen) (D), E15.5 Six1lacZ/+ cochlear (E), E15.5 Six1lacZ/+ cochlear stained with anti-NEUROD1 (red, F). GER and LER are indicated. oc, otic cup; sg, spiral ganglion. Scale bars: 50 μm.
Fig. 3.
Fig. 3.
Co-expression of Eya1 and Six1 induces neuronal phenotypes in cochlear nonsensory epithelial cells. (A,B) Neurod1 whole-mount ISH of explants of E9.25-9.5 embryo transfected with Eya1 and Six1 (green) in electroporated (A) and unelectroporated/control (B) sides. Arrows indicate ectopic neurogenesis in A and normal low level expression in B. (C) Relative mRNA levels of Neurod1 in otocyst including the VIIIth-VIIth ganglion transfected with Eya1 or both Eya1 and Six1 for 1 day. Untransfected side was used as control and Neurod1 mRNA level was designated as 1.0. Data indicate mean ± s.e.m. (D,F) Cochlear explant transfected with Eya1 and Six1 and labeled with anti-TUJ1 (red). The sensory epithelium (SE) and nonsensory GER and LER are indicated. Arrows and arrowheads point to TUJ1+ neurons. In F, higher magnification of the boxed area is shown. (E,G) High-magnification images showing cells transfected with Eya1 and Six1. Arrows point to TUJ1+ cells and arrowheads point to axonal growth cone. Scale bars: 100 μm.
Fig. 4.
Fig. 4.
Eya1 and Six1 induce Neurog1 and Neurod1 expression and are required for neuronal differentiation. (A-C) Cochlear explants transfected with Eya1 and Six1 (green) stained with anti-TUJ (red) and Neurog1 (A) or Neurod1 (B) ISH or anti-NF (red, C). (D) Higher magnification of boxed area in C. (E-H) Cochlear explants transfected with Eya1 and Neurog1 (E), Six1 and Neurog1 (F), Eya1 and Neurod1 (G) or Six1 and Neurod1 (H) and stained for NF.
Fig. 5.
Fig. 5.
Yeast two-hybrid and in situ hybridization analyses. (A) SIX1 or EYA1 domain was used as ‘bait’. pGBKT7 or pACT2 vector alone was used as negative control. Co-transformation was analyzed for ability to activate lacZ expression by liquid β-gal assay. Strength of interactions was judged by the units of β-gal activity. A result representing an example data set of three independent experiments (performed in triplicate) is shown with standard deviation. (B,C) Section ISH for Baf170 and Brg1 in otocyst (ov) and VIIIth ganglion at E10.0 (B) and in developing cochlea (C). Lower panels in C are sections of upper panels and the plane of sections are indicated. oc, organ of Corti; sg, spiral ganglion. Scale bars: 100 μm.
Fig. 6.
Fig. 6.
EYA1 and SIX1 cooperatively interact with BRG1 and BAF170 to drive neuronal differentiation in 3T3 fibroblast cells. (A-H) 3T3 cells were transfected with the indicated constructs (green) and stained with anti-NF (red) (A,C,E,G) or anti-TUJ1 (B,D,F,H). Hoechst stains the nuclei.
Fig. 7.
Fig. 7.
EYA1 and SIX1 cooperatively interact with BAF170 and BRG1 and with BAF155 to drive neurogenesis in cochlear GER cells. (A-D) Explants transfected with Brg1, Eya1 and Six1 (A), Baf170, Eya1 and Six1 (B), or Baf155, Baf170, Brg1, Eya1 and Six1 (C,D) and hybridized with Neurod1 probe and stained with anti-NF (red), anti-GFP (green) and/or anti-MYO7A (green in organ of Corti) (oc). Arrows and open arrowheads point to two ectopic neurons induced in the GER that innervate hair cells.
Fig. 8.
Fig. 8.
BRG1 ATPase activity is necessary for EYA1 and SIX1 to initiate neuronal development and normal neurogenesis in the otocyst. (A-D) 3T3 cells transfected with indicated constructs and stained with anti-GFP (green) for detecting transfected cells and anti-NF (red) for neurons. Hoechst was used for nuclear staining. (E,F) Explants transfected with indicated constructs and stained with anti-NF and anti-GFP. (G-J) Embryos at ∼E9.5 transfected with shBrg1 (G), control shRNA (H), shBrg1, Eya1 and Six1 (I) or unelectroporated (J) and hybridized with Neurod1 probe and stained with anti-GFP. ov, otic vesicle; V-VIII, Vth-VIIIth ganglion. (K) The relative mRNA levels of Neurod1 in the otocyst and VIII/VIIth ganglion transfected with shBrg1, control shRNA and unelectroporated control were quantified by qRT-PCR. Data indicate mean ± s.e.m. P<0.05. Scale bar: 100 μm.
Fig. 9.
Fig. 9.
EYA1 and SIX1 interact directly with BRG1 and BAF170. GST, GST-EYA1 or GST-SIX1 fusion protein was incubated with FLAG-BAF170, FLAG-BRG1, NEUROG1 or NEUROD1 protein and analyzed by western blot using anti-FLAG, anti-NEUROG1 or anti-NEUROD1 antibodies. CoIP of BAF170, BRG1, EYA1, NEUROG1, NEUROD1, SOX2 or SIX1 detected by western blot in SIX1 or HA immunoprecipitates from 3T3 cells transfected with Flag-Baf170, Flag-Brg1, HA-Flag-Eya1, His-Six1 and Sox2. Anti-E2F1 was used as a negative control for detection of the transcription factor E2F1.
Fig. 10.
Fig. 10.
SOX2 cooperates with EYA1 and SIX1 and the SWI/SNF chromatin-remodeling complex to coordinate neuronal differentiation. (A-G) Cochlear explants transfected with the indicated constructs and stained with anti-NF (red) and Neurod1 probe. (H) CoIP of SOX2 and NEUROG1 detected by western blot in SIX1 or HA immunoprecipitates from 3T3 cells transfected with Sox2, Flag-BAF170, Flag-BRG1, HA-Flag-Eya1 and His-Six1. In vitro translated NEUROG1 and SOX2 were loaded as size control.
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