Notch Signal Controls Several Steps of Inner Ear Development Norio Yamamoto and Matthew W

NIDCD Notch signal controls several steps of inner ear development Norio Yamamoto and Matthew W. Kelley Section on Developmental Neuroscience Section on Developmental Neuroscience National Institute on Deafness and Other Communication Disorders National Institutes of Health Abstract Problem addressed RBP-J mutant cochleae did not have any supporting cells Notch signaling has been reported to contribute to inner ear development, however, its specific functions remain unclear, partly because of discrepancies between the phenotypes of mutant mice with single deletion of specific Notch related genes. These discrepancies are probably due to functional compensation by other Notch Figure 4 receptors or ligands. Foxg1 Cre;RBP-J floxed/+ Foxg1 Cre;RBP-J floxed/floxed A B C D To determine the effects of complete elimination of Notch signaling, we used a conditional knockout of the Rbpsuh gene. RBP-J protein is a critical transcriptional p27 Phalloidin p27 Phalloidin To test if mutant cochleae contained supporting cells we co-activator for all Notch molecules and thus deletion of this protein inhibits all Notch signaling. examined expression of supporting cell markers such as p27 and Prox1. On E17.5 p27 was expressed in supporting cells Methods and Measures ***** ***** under or between hair cells such as inner pharyngeal cells, Floxed Rbpsuh mice were crossed with Foxg1-Cre knock-in mice to delete the Rbpsuh gene in the inner ear. Inner ear phenotypes in Rbpsuh conditional knockout Deiter's cells and pillar cells (asterisks in figure 4 A and B). mice were determined at various developmental stages using immunohistochemistry. But no p27 expression was detected in those regions of RBP-J E Prox1 F Phalloidin G Prox1 H Phalloidin conditional knockout mice (Figure 4 C and D). Prox1 was Results expressed in pillar cells and Deiter's cells in control mice The Foxg1-Cre transgene produced variable deletion efficiency of Rbpsuh. When deletion efficiency was low, cochleae contained an increased and decreased (asterisks in figure 4 E and F) but not in mutant mice (Figure 4 numbers of inner and outer hair cells, respectively. These results suggest roles for Notch signaling as a cell-fate determinant. In addition, all vestibular sensory ***** ***** G and H). epithelia were completely absent. When deletion efficiency of Rbpsuh was high, the cochlea contained only a small patch of hair cells located near the apex. Expression of Sox2, a marker of developing sensory epithelia, was reduced. These results suggest that Notch signaling also regulates the induction of inner ear sensory epithelia. Sox2 expression is absent in early cochlear development in RBP-J mutants Conclusions Figure 5 Notch signaling mediates several aspects of inner ear development. Early in inner ear formation, Notch signaling specifies the future sensory epithelia while at later Foxg1 Cre;RBP-J floxed/+ E13.5 whole mount E13.5 whole mount E E17.5 section E17.5 section E17.5 section time points, Notch signaling acts within those epithelia to determine hair or supporting cells. A C G I lateral

Clinical significance of study medial lateral Our results suggest that modulation of the Notch pathway could be used to regenerate hair cells and supporting cells by first specifying sensory epithelia and then modulating cell fates within those epithelia. *** * * *** * * *** * *

Introduction Sox2 medial RBP-J Sox2 Sox2+Phalloidin Myosin6

Notch/RBP-J signaling (Figure 1) Foxg1 Cre;RBP-J floxed/floxed 1. Evolutionally conserved signaling pathway involved in binary cell fate. choice. Recently this signaling pathway has been reported to also B E13.5 whole mount D E13.5 whole mount F E17.5 section H E17.5 section J E17.5 section be involved in maintenance of stem cell populations. 2. In the development of inner ear, hair cell number increases at the expense of supporting cell number when this signaling pathway is disturbed (Lanford et al. 1999, Zine et al. 2000, Kiernan et al. 2005, Takebayashi et al. 2007) 3. The interaction of the Notch receptor and its ligand induces the proteolytic cleavage of the Notch receptor, which requires γ-secretase activity. 4. This cleavage liberates the intracellular domain of Notch (NICD), which then translocates to the nucleus and binds to RBP-J (recombination signal binding protein J), resulting in transcriptional activation of target genes such as Hes1 and Hes5. Sox2 RBP-J F Sox2 Sox2+Phalloidin Myosin6 5. All four of Notch receptors reported in mammals require cleavage by γ-secretase and use one type of RBP-J as a transcriptional We examined Sox2 expression in mutants because it is expressed in cochlear sensory epithelia from an early stage of development and is involved co-activator. Therefore, deletion of the single RBP-J gene in mammals or pharmacological inhibition of γ-secretase activity leads to in development of the sensory domain. At E13.5 expression of Sox2 in mutants was too low to be detected by antibody staining (Figure 5 A and complete inactivation of Notch signaling B). Absence of Sox2 signal correlated with the loss of RBP-J expression (Figure 5 A-D). At E17.5 in control animal Sox2 is expressed weakly in Genetic disruption of RBP-J gene or pharmacological inhibition using γ-secretase inhibitors completely blocks Notch/RBP-J signaling. hair cells (Arrows and an arrowhead in figure 5 E and G) and strongly in supporting cells (asterisks in figure 5 E and G). In mutants, only weak Figure 1 expression of Sox2 was observed (Figure 5 F and H), suggesting these Sox2 positive cells have hair cell characteristics. Myosin 6 staining on ligands (Jagged 1 and 2, Delta 1, 3 and 4) Pharmacological inhibition ligands (Jagged 1 and 2, Delta 1, 3 and 4) adjacent section of cochleae also showed that Sox2 positive cells in mutant cochleae were hair cells (Figure 5 J). of ge vage Notch signaling nt clea These results suggest that Notch signaling regulates Sox2 expression at an early stage of cochlear development but may not play a role in later Notch 4 e dependent cleava Notch 4 e depende Notch 1 Notch 2 Notch 3 γ-secretas Notch 1 Notch 2 Notch 3 γ-secretas regulation of Sox2. The regulation of Sox2 expression in the early stage of cochlear development suggests that Notch signaling is involved in γ-secretase inhibitor induction or maintenance of sensory epithelia in the inner ear. NICD

Hes Math1 cytosol cytosol X X RBP-J Complete deletion of RBP-J leads to small population of inner hair cells located in the apical region of the cochleae RBP-J nucleus Genetic inhibition nucleus of Figure 6 Notch signaling ligands (Jagged 1 and 2, Delta 1, 3 and 4) ABCFoxg1 Cre;RBP-J floxed/+ Foxg1 Cre;RBP-J floxed/floxed Foxg1 Cre;RBP-J floxed/del If Notch signaling regulates Sox2 expression in early

ge stage of cochlear development, no hair cells or Apex Apex Notch 4 e dependent cleava supporting cells should have been observed in RBP-J Notch 2 Notch 3 γ Apex Notch 1 -secretas mutant cochleae because deletion of RBP-J gene inhibits Notch signaling completely. But in Foxg1 NICD Base Base Cre;RBP-J floxed/floxed mice, we observed many inner cytosol X Base hair cells and some outer hair cells in their cochleae RBP-J nucleus Myosin6 Myosin6 Myosin6 (Figures 2 C, 3 and 6 B). We hypothesized this could be DEFoxg1 Cre;RBP-J floxed/del Foxg1 Cre;RBP-J floxed/del F Foxg1 Cre;RBP-J floxed/del Summary of inner ear phenotypes with disruption of various Notch/RBP-J signaling components due to incomplete deletion of the RBP-J gene in Foxg1 Cre;RBP-J floxed/floxed mice. To effect a complete Jag2 KO Jag2/LFNG dKO Notch1 cKO Jag1 cKO Dll1 cKO Cochlea deletion of RBP-J gene, we established an RBP-J IHC # Increased No change Increased Doubled Increased floxed/deletion line and used these mice to produce a OHC # Increased Increased Increased No OHCs Increased Apex Apex Apex more complete deletion of RBP-J gene. In these mice Sox2 n.d. n.d. n.d. Reduced n.d. we observed only a small population of myosin 6 Vestibule Base Base Base Saccule n.d. n.d. n.d. Unaffected Reduced p75 Phalloidin Merged positive hair cells in the apical region located in the Utricle n.d. n.d. n.d. Reduced/no HCs Reduced From Lanford et al. Nat Genet. Mar;21(3):289-92, 1999 cochleae (Figure 6 C). Immunohistochemistry with anti GHIFoxg1 Cre;RBP-J floxed/del Foxg1 Cre;RBP-J floxed/del Foxg1 Cre;RBP-J floxed/del Ampullae n.d. n.d. n.d. Lost or no HCs Less affected p75 antibodies and phalloidin showed that these Lateral Lateral Lateral myosin6 positive hair cells had characteristics of inner hair cells (Figure 6 D-I). Materials and methods

Medial Medial Medial RBP-J floxed mouse (Han et al. 2002) Antibodies used p75 Phalloidin Merged loxP sequences flanked exons 6 and 7 in the RBP-J gene anti Myosin6 antibodies (Proteus Biosciences) 1:1000 anti Sox2 antibodies (Chemicon, AB5603) 1:1000 anti p75 antibodies (Chemicon, AB1503) 1:1000 Pharmacological inhibition of Notch signaling around E12.5 caused small numbers of hair cells Wild type anti S100A1 antibodies (Lab Vision, RB-1800) 1:100 Figure 7 6 7 anti Prox1 antibodies (Covance, PRB-238C-200) 1:2000 SphΣ Sph Σ ABE Floxed anti p27 antibodies (Lab Vision, RB-9019) 1:100 1400 Results from complete deletion of RBP-J gene SphΣ SphΣ anti RBP-J antibodies (Institute of immunology, K0043) 1:150 suggested that Notch signaling has an additional 6 7 Neo 1200 function in the specification of the sensory epithelia SphΣ SphΣ γ-secretase inhibitor treatment from inner ear. In addition, Notch has been shown Foxg1 Cre knock-in mouse (Hebert et al. 2000) 1000 to play a role on specifying cell fate between hair

Cre transgene is expressed specifically in telencephalon, anterior half DAPT (N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl e b and supporting cells. These two functions have ester from Calbiochem, 565784) was used as a γ-secretase inhibitor at 10 µM E12 E14 E16 E18 E12 E14 E16 E18

of optic placode and otic placode under the control of Foxg1 num 800 different time frames; specification of sensory

promoter. Otic expression begins at E9.0.

D C a epithelia occurs around E12 and specification of cell

ot t 600 fate occurs around E14. To confirm this we used DAPT to transiently inhibit g-secretase and, 400 Results therefore, Notch activity at different times between 200 E12 and E18. Inhibition from E12-E14 (Figure 7 B) caused small numbers of hair cells (Figure 6 E) E12 E14 E16 E18 E12 E14 E16 E18 Genetic disruption of RBP-J results in short cochleae RBP-J mutant cochleae showed increased numbers of inner 0 as compared with control or other treatment Control E12-14E14-16 E16-18 and a lack of vestibular organs in inner ears hair cells and loss of most outer hair cells conditions. This result suggests that Notch signaling specifies sensory epithelia between E12-E14. Figure 2 Figure 3 A Foxg1 Cre;RBP-J floxed/+ Foxg1 Cre;RBP-J floxed/floxed Foxg1 Cre;RBP-J floxed/+ Foxg1 Cre;RBP-J floxed/floxed A Myosin6 B Myosin6 C Phalloidin D Myosin6 + Phalloidin lateral No hair cells exist in vestibular organs of RBP-J mutant mice Foxg1 Cre;RBP-J floxed/floxed OHC Figure 8 IHC Crista ampullaris Saccular macula Utricular macula * * Myosin6 From macroscopic analysis (Figure 1 A), vestibular organs seemed not to develop in RBP-J mutant mice. Analysis of E p75 F p75 G Phalloidin H p75 + Phalloidin section of vestibular organs (Figure 8) showed that vestibular sensory epithelia did not develop in RBP-J mutant mice

OHC because no myosin 6 positive cells were detected by IHC E17.5 inner ear immunohistochemistry. Phalloidin staining showed that vestibular organs themselves developed in mutant mice. B Foxg1 Cre;RBP-J floxed/+C Foxg1 Cre;RBP-J floxed/floxed I S100A1 J S100A1 K Phalloidin L S100A1 + Phalloidin These results also support a role of Notch signaling in Phalloidin specification of sensory epithelia in the inner ear.

Apex OHC Apex IHC

medial Base Base Myosin6 staining on middle turns of E17.5 cochleae (Figure 3A-D) suggests that only inner hair cells exist in this region. To characterize these hair cells, we stained E17.5 cochlea, Myosin6 staining E17.5 cochlea, Myosin6 staining cochlear epithelia with antibodies against p75 (Figure 3E-H, marker for pillar cells Vestibular organs including crista ampullaris (arrow heads in figure that exist between inner and outer hair cells) and S100A1 (Figure 3 I-L, positive Discussion and Conclusions 2A), saccule (arrows in figure 2A) and utricle (asterisks in figure 2A) strongly in inner hair cells and weakly in Deiter's cells, supporting cells located 1. are absent in Foxg1 Cre;RBP-J floxed/floxed mice. under outer hair cells) at the same stage. Notch signaling mediates several steps of inner ear development. Early in inner ear formation, Notch signaling specifies the future Cochlear length is shorter in Foxg1 Cre;RBP-J floxed/floxed mice Myosin6 positive hair cells in RBP-J mutants are medial to p75 positive cells sensory epithelia while at later time points, Notch signaling acts within those epithelia to determine hair or supporting cells. This (Figure 2B and C). Visualization of hair cells with anti Myosin6 (Figure 3 F-H) and strongly positive for S100A1 (Figure 3 J-L), suggesting that is supported by results from both genetic and pharmacological inhibition of Notch signaling. antibodies indicates an increased number of inner, but no outer hair these cells are inner hair cells. 2. cells in the basal and middle turns of cochleae. A small numbers of Our results suggest that modulation of the Notch pathway could be used to regenerate hair cells and supporting cells by first outer hair cells were present in the apex (Figure 2C) at E17.5. specifying sensory epithelia and then modulating cell fates within those epithelia.