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First published online 16 August 2006
doi: 10.1242/dev.02536

Development 133, 3683-3693 (2006)
Published by The Company of Biologists 2006
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COUP-TFI controls Notch regulation of hair cell and support cell differentiation

Louisa S. Tang, Heather M. Alger* and Fred A. Pereira{dagger}

Huffington Center on Aging, Department of Otolaryngology - Head and Neck Surgery, Department of Molecular and Cellular Biology, Program in Cell and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.


Figure 1
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  		Fig. 1. COUP-TFI-/- cochlea exhibit supernumerary hair cells and Deiter's supporting cells. (A) Flat-mounts (a-f) and cross-sections (g-j) of wild-type (a,c,e,g,i) and COUP-TFI-/- (b,d,f,h,j) cochlear duct at P10, stained with Alexa 488-conjugated phalloidin (a-f), anti-myosin VIIa (g,h), and Hematoxylin and Eosin (H&E, i,j). Extra inner hair cells (IHC) were seen in the basal (compare b with a) and apical (compare f with e) regions, and extra outer hair cells (OHC) were seen in the middle (compare numbered rows in d with c) and apical (compare rows in f with e) regions of COUP-TFI-/- cochlea. OHC stereociliary bundles were often misoriented in COUP-TFI-/- cochlea (direction of arrows is perpendicular to the kinocilium in f). (g,h) Cross-sections through the apical region of the cochlear duct show the typical pattern of one row of IHC and three rows of OHC expressing myosin VIIa (red) in the wild type, but the COUP-TFI-/- mutant has supernumerary hair and support cells (arrowheads). Nuclei were counterstained with DAPI (blue). (i,j) H&E-stained sections of apical regions show individual supporting cells underlying every hair cell (arrowheads, compare j with i). Scale bar: 15 µm. (B) Paint-filling of wild-type (a,c,e) and COUP-TFI-/- (b,d,f) cochlear ducts at E13, E15 and E17 show that COUP-TFI-/- mutants have a shorter cochlear duct. Scale bar: 100 µm. (C) Graph of hair cell counts (IHC and OHC) from different regions of the cochlear duct at P10, showing more hair cells in the apical region of the COUP-TFI-/- duct than in the wild type (*P<0.05).

 

Figure 2
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  		Fig. 2. COUP-TFI-/- sensory epithelium exhibited extra proliferating cells and ectopic hair cell differentiation in the supporting cell region. (A-H) Immunofluorescence detection of p27 (red) to mark the zone of non-proliferation (ZNP) in the sensory epithelia in wild-type (A,C,E,G) and COUP-TFI-/- (B,D,F,H) cochlea at E15.5 (A-F) and E17.5 (G,H). The ZNP was similar at the basal and middle cochlear turns (A-D). However, the ZNP domain at the apex was widened (compare double arrows in E with F) and encompassed extra hair cells (compare G with H) in the COUP-TFI-/- mutants (myosin VIIa, green; GFI lectin I, blue). The onset of hair cell differentiation was unchanged in the COUP-TFI-/- basal (A,B) and apical (E,F) regions (arrowheads point to myosin VIIa-positive cells). (I,J) BrdU labeling (green) in wild-type (I) and COUP-TFI-/- (J) epithelia at E16.5 shows extra positive cells in the outer sulcus region of the apical turn in the COUP-TFI-/- mutants (arrows). Sections were co-localized with myosin VIIa (red, arrowheads). (K,L) p27 (green) expression in adjacent sections labeled with BrdU, confirmed the BrdU-positive cells in outer sulcus region were outside the ZNP. (M-P) Immunolocalization of Ki67 in wild-type (M,O) and COUP-TFI-/- (N,P) cochlea at E16.5 (M,N) and P0 (O,P). Proliferating cells were found in the outer sulcus region at E16.5 (red) and inner sulcus region at P0 (green) in the apical turn of COUP-TFI-/- mutants (arrows). (Q) Quantification of BrdU-positive and Ki67-positive cells at E16.5. (R-T) Deconvolution microscopy images show ectopic expression of myosin VIIa (asterisks in S and T) in the supporting cell compartment of the COUP-TFI-/- duct at P0. Scale bars: in A-P, 40 µm; in R-T, 100 µm.

 

Figure 3
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  		Fig. 3. Deregulation of Notch signaling molecules in COUP-TFI-/- cochlea. (A-C') In situ hybridization on sections of wild-type (A-D,I-L,Q-R,V-Y) and COUP-TFI-/- (E-H,M-P,T,U,Z-C') cochlea for Jag1 (A-H), Hes5 (I-P), and Lfng (Q,R,T-C'). (A-D) At E15.5, Jag1 in wild type was predominately expressed in the region where the non-sensory cells (arrow) are differentiating. (E-H) In COUP-TFI-/- cochlea, there was a lack of upregulation of Jag1 in the supporting cells region. (I-P) Hes5 expression in wild type (I-L) was restricted to the Deiter's supporting cells (SC) of the sensory epithelium, whereas in COUP-TFI-/- mutants (M-P), Hes5 expression was downregulated. (Q,R,T-C') At E14.5, there was a dramatic expansion in the Lfng expression domain (compare R and U, circles); at E15.5, expansion to the greater epithelial ridge (GER) was observed only in the wild type (W-Y), whereas expansion to the lesser epithelial ridge (LER) was also observed in COUP-TFI-/- mutants (A'-C'). (S) Immunofluorescence staining of COUP-TFI ({alpha}-COUP-TFI) in wild-type sensory epithelium at E14.5 (arrowhead marks a hair cell). Scale bar: in A,E,I,M,Q,T,V,Z, 200 µm; in B-D,F-H,J-L,N-P,R,S,U,W-Y,A'-C', 50 µm.

 

Figure 4
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  		Fig. 4. DAPT treatment of cochleae in culture induces hair cell differentiation. (A) Real-time PCR analysis of myosin VIIa transcripts is presented in terms of average {Delta}Ct (see Materials and methods). A greater Ct value represents a lower transcript expression level. Histogram and bars represent average {Delta}Ct±s.d. (*P<0.05). High DAPT doses increase myosin VIIa expression, with significant induction at 5 µM DAPT. (B) Western blot confirmation of myosin VIIa protein expression changes with DAPT dose. The myosin band of Kaleidoscope prestained standard is used as control. (C) Expression of COUP-TFI and COUP-TFII transcripts in cochleae treated with DAPT. COUP-TFI and COUP-TFII expression was not changed with DAPT, even at the highest concentration of 5 µM. Because DAPT can induce hair cell differentiation (in terms of myosin VIIa expression as shown in Fig. 4A), the value is presented here after further normalization with the myosin VIIa values. (D) Expression of Hes5 in cochleae after culture with DAPT. Hes5 expression is reduced with increasing concentrations of DAPT, with statistically significant differences at 2.5 and 5 µM (*P<0.05). A statistically significant reduction was also found between COUP-TFI-/- and wild type at 5 µM (**P<0.05).

 

Figure 5
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  		Fig. 5. Quantification of hair cell differentiation and rows in wild-type and COUP-TFI-/- cochlea after DAPT treatment. (A) Real-time PCR analysis of myosin VIIa transcripts shows hair cell differentiation is dependent on DAPT dose. There is a significant difference in the COUP-TFI-/- compared with wild type at the concentration of 5 µM (**P<0.05). (B) Western blot confirmation of myosin VIIa protein expression after treatment with 5 µM DAPT shows an increase with DAPT treatment. The myosin band of Kaleidoscope prestained standard is used as control. (C) Hair cell row counts (myosin VIIa-positive inner and outer hair cells) along the length of the cochlear duct after culture with 5 µM DAPT. Both wild-type and COUP-TFI-/- cochleae showed significant increases in hair cell rows after DAPT treatment (*P<0.05), and COUP-TFI-/- mutants had a significantly greater number than wild type in the apex (**P<0.05). (D) Immunostained sections of DAPT-treated cochleae at the apical region show an increase in hair cell rows in the COUP-TFI-/- mutants (c,d) compared with wild type (a,b; note, image in D part d is an extreme case of extra hair cells). Sections show hair cells expressing myosin VIIa (red) and nuclei counterstained with DAPI (blue). Scale bar: 20 µm.

 

Figure 6
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  		Fig. 6. A model of the functional hierarchy of COUP-TFI modulation of Notch signaling in hair cell and support cell differentiation. In the wild type, Lfng modulates Notch activities by limiting Notch ligand-receptor interactions. This process is regulated by the restricted expression of Lfng that allows strong binding of Notch with ligands such as Jag1, which promotes Notch signaling and the expression of downstream target genes, such as Hes5. Hes5 inhibits the expression of Math1, leading to a normal limit of hair cell and support cell differentiation. COUP-TFI may directly modulate these Notch components in regulating hair cell and support cell differentiation. The absence of COUP-TFI results in expansion of the Lfng expression domain and an increasing inhibition of Notch ligand-receptor interactions, and results in a reduced expression of Hes5. Reduced Hes5 levels have a less inhibitory effect on Math1 transcription, which allows Math1 to induce extra hair cell differentiation.

 

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© The Company of Biologists Ltd 2006