The ATP-dependent chromatin remodeling enzyme CHD7 regulates pro-neural gene expression and neurogenesis in the inner ear - PubMed (original) (raw)

The ATP-dependent chromatin remodeling enzyme CHD7 regulates pro-neural gene expression and neurogenesis in the inner ear

Elizabeth A Hurd et al. Development. 2010 Sep.

Abstract

Inner ear neurogenesis is positively regulated by the pro-neural bHLH transcription factors Ngn1 and NeuroD, but the factors that act upstream of this regulation are not well understood. Recent evidence in mouse and Drosophila suggests that neural development depends on proper chromatin remodeling, both for maintenance of neural stem cells and for proper neuronal differentiation. Here, we show that CHD7, an ATP-dependent chromatin remodeling enzyme mutated in human CHARGE syndrome, is necessary for proliferation of inner ear neuroblasts and inner ear morphogenesis. Conditional deletion of Chd7 in the developing otocyst using Foxg1-Cre resulted in cochlear hypoplasia and complete absence of the semicircular canals and cristae. Conditional knockout and null otocysts also had reductions in vestibulo-cochlear ganglion size and neuron number in combination with reduced expression of Ngn1, Otx2 and Fgf10, concurrent with expansion of the neural fate suppressor Tbx1 and reduced cellular proliferation. Heterozygosity for Chd7 mutations had no major effects on expression of otic patterning genes or on cell survival, but resulted in decreased proliferation within the neurogenic domain. These data indicate that epigenetic regulation of gene expression by CHD7 must be tightly coordinated for proper development of inner ear neuroblasts.

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Figures

Fig. 1.

Generation of a conditional Chd7 allele. (A) Recombineering techniques were used to introduce a neomycin/kanamycin (N/k) cassette downstream of Chd7 exon 2. LoxP sites (grey arrowheads) were positioned 5′ of exon 2 and 3′of the N/k cassette. (B) Targeted ES cells were confirmed by Southern blotting, using probes indicated in A. Genomic DNA was digested with _Eco_RI (R) producing either a 12 kb wild-type (WT) or 9.9 kb targeted (T) product, whereas digestion with _Bam_HI (B) and screening with a 3′ probe detected either a 13.1 kb (WT) or 10.8 kb targeted (T) product. (C) PCR genotyping of DNA from EIIa-Cre;Chd7+/flox mice. Bands of 1249, 987 and 324 bp represent Chd7+, Chd7flox and Chd7− alleles, respectively. (D) CHD7 immunofluorescence on E10.5 sagittal frozen sections. Chd7+/− embryos have reduced Chd7 expression compared with Chd7+/+, whereas CHD7 protein is absent in Chd7−/− embryos. White lines in D represent the outline of the otic epithelium. Scale bar: 100 μm.

Fig. 2.

Foxg1-Cre;Chd7Gt/flox mice have severe inner ear abnormalities. (A) Breeding scheme used to generate mice with conditional loss of Chd7 function within the inner ear. (B-D) CHD7 is widely expressed in the E10.5 otocyst of Foxg1-Cre;Chd7+/flox (B) and Chd7+/flox (C) embryos, and is absent from the Foxg1-Cre;Chd7Gt/flox otocyst (D). Sections are in the transverse plane, with dorsal towards the top, medial towards the left, and the hindbrain epithelium is on the left, as shown by arrows in B. (E,F) Chd7Gt/flox and Foxg1-Cre;Chd7+/flox E14.5 inner ears have normal anterior and posterior semicircular canals, endolymphatic ducts and cochleae. Inserts in E and F are enlarged images of typical lateral canal defects, including epithelial narrowing (arrow) and truncation (arrowhead). (G) Foxg1-Cre;Chd7Gt/flox E14.5 ears have no semicircular canals or sensory vestibular structures aside from a rudimentary anterior semicircular canal (arrow) and an undercoiled cochlea with abnormal twisting at the tip of the cochlea (*). The endolymphatic duct (ed) is intact but smaller in Foxg1-Cre;Chd7Gt/flox ears. (H-J) Staining of P0 inner ears shows strong β-galactosidase expression in Chd7Gt/flox vestibular sensory organs [anterior ampulla (aa), lateral ampulla (la), posterior ampulla (pa), saccule (sac) and utricle (ut)] and cochlea (coc). (J) Foxg1-Cre;Chd7Gt/flox ears have β-galactosidase staining in the abnormally developed cochlea. Asterisk indicates abnormal twisting at the cochlear tip. Other abbreviations: asc, anterior semicircular canal; lsc, lateral semicircular canal; psc, posterior semicircular canal. Scale bar: 100 μm.

Fig. 3.

Domain-specific patterning in the developing otocyst is sensitive to Chd7 dose_._ All sections are in the transverse plane with dorsal towards the top and lateral towards the right, as shown in A. A-DD are E10.5; EE-GG,II,JJ are E9.5; HH is E9.0. (A-E) Expression of the ventral otocyst marker Otx2 is unchanged in Chd7 Gt/+ (B) and Foxg1-Cre;Chd7+/flox (D) otocysts compared with wild type, but is absent from Chd7Gt/Gt (C) and Foxg1-Cre;Chd7Gt/flox (E) otocysts. (F-J) Ventral Lfng expression is conserved in all Chd7 mutant otocysts, albeit at lower levels in Chd7Gt/Gt (H). (K-O) Eya1 is expressed in Chd7 Gt/+ (L), Foxg1-Cre;Chd7+/flox (N) and Foxg1-Cre;Chd7Gt/flox (O) otocysts but is absent from Chd7Gt/Gt (M). (P-T) Fgf10 is expressed in Chd7 Gt/+ (Q) and Foxg1-Cre;Chd7+/flox (S) otocysts, and is absent from Chd7Gt/Gt (R) and Foxg1-Cre;Chd7Gt/flox (T). (U-Y) TBX1 protein is expanded ventrally in Chd7Gt/Gt (W) and Foxg1-Cre;Chd7Gt/flox (Y) otocysts compared with wild type (U) and Chd7 Gt/+ (V). (Z-JJ) The Ngn1 expression domain is maintained in E9.5 and E10.5 Chd7 Gt/+ (AA,FF) and Foxg1-Cre;Chd7+/flox otocysts (CC,II), is absent from E9.5 and E10.5 Chd7Gt/Gt (BB,GG) and is reduced in Foxg1-Cre;Chd7Gt/flox otocysts (DD,JJ). (HH) Ngn1 expression is reduced but present in E9.0 Chd7Gt/Gt. Scale bar: 100 μm.

Fig. 4.

CHD7 is concentrated in early inner ear neuroblasts of the vestibulo-cochlear ganglion. Co-immunofluorescence of anti-CHD7 (A,D,G,J), with anti-β-tubulin III (TuJ1) (B), NeuroD (E) or Islet1 (H) shows doubly positive cells throughout the otocyst and ganglia of E10.5 wild-type embryos. (C,F,I) Merged images. Mitotic cells within the ventral otocyst and ganglion are also CHD7 positive, as shown by co-localization (L) with anti-phosphohistone H3 (K). Arrow in F represents CHD7-positive, NeuroD-negative cells. Scale bar: 100 μm.

Fig. 5.

Chd7 germline and conditional mutants have reduced inner ear neuroblasts. (A-D) TuJ1 expression and vestibulo-cochlear ganglia size are reduced in E10.5 Chd7 Gt/+ (B), Chd7Gt/Gt (C) and Foxg1-Cre;Chd7Gt/flox (D) mutants. (E-H) Islet1 positive neuroblasts in the vestibulo-cochlear ganglion are reduced in Chd7 Gt/+ (F), Chd7Gt/Gt (G) and Foxg1-Cre;Chd7Gt/flox (H) embryos. Sections are in the transverse plane, with dorsal towards the top and lateral towards the right as shown by arrows in A. (I) Isl1+ cells are reduced in the ganglion of Chd7 Gt/+ (dark blue), Chd7Gt/Gt (black), Foxg1-Cre;Chd7+/flox (light blue) and Foxg-Cre;Chd7Gt/flox (red) as compared with wild type (white). Scale bar: 100 μm. ***_P_≤0.001.

Fig. 6.

Neuroblasts are reduced in both epithelium and ganglion of Chd7 mutants. (A-I) Sections are in the transverse plane, with dorsal towards the top and lateral towards the right as shown in A. (A,B,J,K) NeuroD-positive cells are unchanged in the E9.5 Chd7 Gt/+ (B) otic epithelium versus wild-type embryos (A) but decreased in the vestibulo-cochlear ganglion. (A,C,D,F,J,K) NeuroD-positive neuroblasts are significantly reduced in both the epithelium and ganglion of E9.5 and E10.5 Chd7Gt/Gt embryos (C,F) relative to wild type (A,D). (D,E,J,K) NeuroD-positive neuroblasts are decreased in both the epithelium and ganglion of E10.5 Chd7 Gt/+ (E) compared with wild type (D). (G-I,J,K) The number of NeuroD-positive cells in the E11.5 epithelium and ganglion is similar in Chd7+/+ (G), Chd7 Gt/+ (H) and Foxg1-Cre;Chd7+/flox (J,K), whereas the number of NeuroD-positive cells in Foxg1-Cre;Chd7Gt/flox (I) is significantly decreased. (J,K)*_P_≤0.05, **_P_≤0.005, ***_P_=0.001. Scale bar: 100 μm. White lines in A-I delimit the otocyst epithelium.

Fig. 7.

CHD7 regulation of inner ear cellular proliferation is dose and tissue dependent. (A,B) Significant decreases in numbers of H3-positive cells are noted in the epithelium and ganglion of Chd7Gt/Gt (black) and Foxg1-Cre;Chd7Gt/flox (red) embryos compared with controls (white). (C-F) Adjacent sections of E10.5 Chd7+/+ (C,E) and Chd7 Gt/+ (D,F) embryos show Ngn1 mRNA (C,D) and anti-phosphohistone H3 (E,F). Sections are in the transverse plane, with dorsal towards the top and lateral towards the right. Scale bar in C is 100 μm and applies to C-F. (G) H3-positive cells are decreased in number in the neurogenic domain of E9.5 and E10.5 Chd7 Gt/+ embryos (blue) compared with wild-type littermates (white). *_P_≤0.01; **_P_≤0.005; *** _P_≤0.001.

Fig. 8.

Model of CHD7 function in genetic cascades that regulate inner ear neurogenesis based on our expression data and on published reports. Shown on the left are proposed genetic cascades in wild type, with changes in Chd7 mutants on the right. Solid and broken lines represent positive and negative regulation of downstream genes, respectively. Genes in grey, red or blue illustrate absent, reduced or increased expression in Chd7 mutant embryos, respectively. CHD7 activates, either directly or indirectly, expression of FGF10 and NGN1, which results in fewer delaminated NeuroD+, ISL1+ and β-tubulin III (TUJ1)+ neuroblasts. EYA1 also regulates this neurogenic cascade, potentially via CHD7-independent mechanisms. Both CHD7 and EYA1 negatively regulate TBX1, which is an inhibitor of NGN1 and neurogenesis. Like TBX1 and EYA1, CHD7 positively regulates OTX2, which is crucial for cochlear development. LFNG is activated by EYA1 and inhibited by TBX1, but is not sensitive to CHD7 dose.

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The ATP-dependent chromatin remodeling enzyme CHD7 regulates pro-neural gene expression and neurogenesis in the inner ear - PubMed (original) (raw)