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

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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.
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