The Notch ligand JAG1 is required for sensory progenitor development in the mammalian inner ear

In mammals, six separate sensory regions in the inner ear are essential for hearing and balance function. Each sensory region is made up of hair cells, which are the sensory cells, and their associated supporting cells, both arising from a common progenitor. Little is known about the molecular mechanisms that govern the development of these sensory organs. Notch signaling plays a pivotal role in the differentiation of hair cells and supporting cells by mediating lateral inhibition via the ligands Delta-like 1 and Jagged (JAG) 2. However, another Notch ligand, JAG1, is expressed early in the sensory patches prior to cell differentiation, indicating that there may be an earlier role for Notch signaling in sensory development in the ear. Here, using conditional gene targeting, we show that the Jag1 gene is required for the normal development of all six sensory organs within the inner ear. Cristae are completely lacking in Jag1-conditional knockout (cko) mutant inner ears, whereas the cochlea and utricle show partial sensory development. The saccular macula is present but malformed. Using SOX2 and p27^kip1 as molecular markers of the prosensory domain, we show that JAG1 is initially expressed in all the prosensory regions of the ear, but becomes down-regulated in the nascent organ of Corti by embryonic day 14.5, when the cells exit the cell cycle and differentiate. We also show that both SOX2 and p27^kip1 are down-regulated in Jag1-cko inner ears. Taken together, these data demonstrate that JAG1 is expressed early in the prosensory domains of both the cochlear and vestibular regions, and is required to maintain the normal expression levels of both SOX2 and p27^kip1. These data demonstrate that JAG1-mediated Notch signaling is essential during early development for establishing the prosensory regions of the inner ear.     

Emx2 and early hair cell development in the mouse inner ear

Emx2 is a homeodomain protein that plays a critical role in inner ear development. Homozygous null mice die at birth with a range of defects in the CNS, renal system and skeleton. The cochlea is shorter than normal with about 60% fewer auditory hair cells. It appears to lack outer hair cells and some supporting cells are either absent or fail to differentiate. Many of the hair cells differentiate in pairs and although their hair bundles develop normally their planar cell polarity is compromised. Measurements of cell polarity suggest that classic planar cell polarity molecules are not directly influenced by Emx2 and that polarity is compromised by developmental defects in the sensory precursor population or by defects in epithelial cues for cell alignment. Planar cell polarity is normal in the vestibular epithelia although polarity reversal across the striola is absent in both the utricular and saccular maculae. In contrast, cochlear hair cell polarity is disorganized. The expression domain for Bmp4 is expanded and Fgfr1 and Prox1 are expressed in fewer cells in the cochlear sensory epithelium of Emx2 null mice. We conclude that Emx2 regulates early developmental events that balance cell proliferation and differentiation in the sensory precursor population.     

Notch ligands with contrasting functions: Jagged1 and Delta1 in the mouse inner ear

Each of the sensory patches in the epithelium of the inner ear is a mosaic of hair cells and supporting cells. Notch signalling is thought to govern this pattern of differentiation through lateral inhibition. Recent experiments in the chick suggest, however, that Notch signalling also has a prior function - inductive rather than inhibitory - in defining the prosensory patches from which the differentiated cells arise. Several Notch ligands are expressed in each patch, but their individual roles in relation to the two functions of Notch signalling are unclear. We have used a Cre-LoxP approach to knock out two of these ligands, Delta1 (Dll1) and Jagged1 (Jag1), in the mouse ear. In the absence of Dll1, auditory hair cells develop early and in excess, in agreement with the lateral inhibition hypothesis. In the absence of Jag1, by contrast, the total number of these cells is strongly reduced, with complete loss of cochlear outer hair cells and some groups of vestibular hair cells, indicating that Jag1 is required for the prosensory inductive function of Notch. The number of cochlear inner hair cells, however, is almost doubled. This correlates with loss of expression of the cell cycle inhibitor p27(Kip1) (Cdkn1b), suggesting that signalling by Jag1 is also needed to limit proliferation of prosensory cells, and that there is a core part of this population whose prosensory character is established independently of Jag1-Notch signalling. Our findings confirm that Notch signalling in the ear has distinct prosensory and lateral-inhibitory functions, for which different ligands are primarily responsible.     

Notch signaling during cell fate determination in the inner ear

In the inner ear, Notch signaling has been proposed to specify the sensory regions, as well as regulate the differentiation of hair cells and supporting cell within those regions. In addition, Notch plays an important role in otic neurogenesis, by determining which cells differentiate as neurons, sensory cells and non-sensory cells. Here, I review the evidence for the complex and myriad roles Notch participates in during inner ear development. A particular challenge for those studying ear development and Notch is to decipher how activation of a single pathway can lead to different outcomes within the ear, which may include changes in the intrinsic properties of the cell, Notch modulation, and potential non-canonical pathways.     

KSHV Manipulates Notch Signaling by DLL4 and JAG1 to Alter Cell Cycle Genes in Lymphatic Endothelia

Increased expression of Notch signaling pathway components is observed in Kaposi sarcoma (KS) but the mechanism underlying the manipulation of the canonical Notch pathway by the causative agent of KS, Kaposi sarcoma herpesvirus (KSHV), has not been fully elucidated. Here, we describe the mechanism through which KSHV directly modulates the expression of the Notch ligands JAG1 and DLL4 in lymphatic endothelial cells. Expression of KSHV-encoded vFLIP induces JAG1 through an NFκB-dependent mechanism, while vGPCR upregulates DLL4 through a mechanism dependent on ERK. Both vFLIP and vGPCR instigate functional Notch signalling through NOTCH4. Gene expression profiling showed that JAG1- or DLL4-stimulated signaling results in the suppression of genes associated with the cell cycle in adjacent lymphatic endothelial cells, indicating a role for Notch signaling in inducing cellular quiescence in these cells. Upregulation of JAG1 and DLL4 by KSHV could therefore alter the expression of cell cycle components in neighbouring uninfected cells during latent and lytic phases of viral infection, influencing cellular quiescence and plasticity. In addition, differences in signaling potency between these ligands suggest a possible complementary role for JAG1 and DLL4 in the context of KS.     

FGF信号通路在内耳发育调控和毛细胞再生中的作用

内耳是感受听觉和平衡觉的复杂器官。在内耳发育过程中,成纤维生长因子(fibroblast growth factor, FGF)信号通路参与了听基板的诱导、螺旋神经节(statoacoustic ganglion, SAG)的发育以及Corti器感觉上皮的分化。FGF信号开启了内耳早期发育的基因调控网络,诱导前基板区域以及听基板的形成。正常表达的FGF信号分子可促进听囊腹侧成神经细胞的特化,但成熟SAG神经元释放的过量FGF5可抑制此过程,形成负反馈环路使SAG在稳定状态下发育。FGF20在Notch信号通路的调控下参与了前感觉上皮区域向毛细胞和支持细胞的分化过程,而内毛细胞分泌的FGF8可调控局部支持细胞分化为柱细胞。人类FGF信号通路异常可导致多种耳聋相关遗传病。此外,FGF信号通路在低等脊椎动物毛细胞自发再生以及干细胞向内耳毛细胞诱导过程中都起到了关键作用。本文综述了FGF信号通路在内耳发育调控以及毛细胞再生中的作用及其相关研究进展,以期为毛细胞再生中FGF信号通路调控机制的阐明奠定理论基础。     

Overactivation of Notch1 signaling induces ectopic hair cells in the mouse inner ear in an age-dependent manner

Background

During mouse inner ear development, Notch1 signaling first specifies sensory progenitors, and subsequently controls progenitors to further differentiate into either hair cells (HCs) or supporting cells (SCs). Overactivation of NICD (Notch1 intracellular domain) at early embryonic stages leads to ectopic HC formation. However, it remains unclear whether such an effect can be elicited at later embryonic or postnatal stages, which has important implications in mouse HC regeneration by reactivation of Notch1 signaling.

Methodology/Principal Findings

We performed comprehensive in vivo inducible overactivation of NICD at various developmental stages. In CAG^CreER+; Rosa26-NICD^loxp/+ mice, tamoxifen treatment at embryonic day 10.5 (E10.5) generated ectopic HCs in the non-sensory regions in both utricle and cochlea, whereas ectopic HCs only appeared in the utricle when tamoxifen was given at E13. When tamoxifen was injected at postnatal day 0 (P0) and P1, no ectopic HCs were observed in either utricle or cochlea. Interestingly, Notch1 signaling induced new HCs in a non-cell-autonomous manner, because the new HCs did not express NICD. Adjacent to the new HCs were cells expressing the SC marker Sox10 (either NICD+ or NICD-negative).

Conclusions/Significance

Our data demonstrate that the developmental stage determines responsiveness of embryonic otic precursors and neonatal non-sensory epithelial cells to NICD overactivation, and that Notch 1 signaling in the wild type, postnatal inner ear is not sufficient for generating new HCs. Thus, our genetic mouse model is suitable to test additional pathways that could synergistically interact with Notch1 pathway to produce HCs at postnatal ages.     

The role of Math1 in inner ear development: Uncoupling the establishment of the sensory primordium from hair cell fate determination

During embryonic development of the inner ear, the sensory primordium that gives rise to the organ of Corti from within the cochlear epithelium is patterned into a stereotyped array of inner and outer sensory hair cells separated from each other by non-sensory supporting cells. Math1, a close homolog of the Drosophila proneural gene atonal, has been found to be both necessary and sufficient for the production of hair cells in the mouse inner ear. Our results indicate that Math1 is not required to establish the postmitotic sensory primordium from which the cells of the organ of Corti arise, but instead is limited to a role in the selection and/or differentiation of sensory hair cells from within the established primordium. This is based on the observation that Math1 is only expressed after the appearance of a zone of non-proliferating cells that delineates the sensory primordium within the cochlear anlage. The expression of Math1 is limited to a subpopulation of cells within the sensory primordium that appear to differentiate exclusively into hair cells as the sensory epithelium matures and elongates through a process that probably involves radial intercalation of cells. Furthermore, mutation of Math1 does not affect the establishment of this postmitotic sensory primordium, even though the subsequent generation of hair cells is blocked in these mutants. Finally, in Math1 mutant embryos, a subpopulation of the cells within the sensory epithelium undergo apoptosis in a temporal gradient similar to the basal-to-apical gradient of hair cell differentiation that occurs in the cochlea of wild-type animals.     

Suppression of Th2 cell development by Notch ligands Delta1 and Delta4

Notch signaling plays important roles in Th cell activation. We show that in response to TLR ligation, dendritic cells up-regulate expression of Notch ligands Delta1 and Delta4 via a MyD88-dependent pathway. Expression of Delta1 or Delta4 by dendritic cells enhanced their ability to activate naive Th cells and promote Th1 cell development, and allowed them to strongly inhibit Th2 cell development. Promotion of Th1 cell development was dependent on IFN-gamma and T-bet expression by responding Th cells. However, the inhibition of Th2 cell development occurred independently of IFN-gamma or T-bet, and resulted from a block in IL-4-initiated commitment to the Th2 lineage. The promotion of Th1 cell development by Delta is not a reflection of the delivery of pro-Th1 instructional signal, but rather it is the result of a block in the downstream effects initiated by IL-4 signaling.     

Notch2 and Notch3 function together to regulate vascular smooth muscle development

Notch signaling has been implicated in the regulation of smooth muscle differentiation, but the precise role of Notch receptors is ill defined. Although Notch3 receptor expression is high in smooth muscle, Notch3 mutant mice are viable and display only mild defects in vascular patterning and smooth muscle differentiation. Notch2 is also expressed in smooth muscle and Notch2 mutant mice show cardiovascular abnormalities indicative of smooth muscle defects. Together, these findings infer that Notch2 and Notch3 act together to govern vascular development and smooth muscle differentiation. To address this hypothesis, we characterized the phenotype of mice with a combined deficiency in Notch2 and Notch3. Our results show that when Notch2 and Notch3 genes are simultaneously disrupted, mice die in utero at mid-gestation due to severe vascular abnormalities. Assembly of the vascular network occurs normally as assessed by Pecam1 expression, however smooth muscle cells surrounding the vessels are grossly deficient leading to vascular collapse. In vitro analysis show that both Notch2 and Notch3 robustly activate smooth muscle differentiation genes, and Notch3, but not Notch2 is a target of Notch signaling. These data highlight the combined actions of the Notch receptors in the regulation of vascular development, and suggest that while these receptors exhibit compensatory roles in smooth muscle, their functions are not entirely overlapping.     

Expression of neurite outgrowth factor and gicerin during inner ear development and hair cell regeneration in the chick

Several cell adhesion molecules are expressed in the developing inner ear. The present study focused on gicerin, a novel member of the immunoglobulin superfamily, in an attempt to improve our understanding of the development and regeneration of chick inner ear. Gicerin is known to homophilically interact with itself and to bind to neurite outgrowth factor (NOF). The data collected herein show that gicerin is highly expressed in auditory epithelium and acoustic ganglion during early embryogenesis. The immunoreactivity of gicerin in the auditory epithelium decreases more rapidly than that in the acoustic ganglion as the mature hair cells become distinguishable. At the post-hatch stage, the expression of gicerin is not observed. In contrast, NOF was expressed on the basement membranes around the auditory epithelium, and in the acoustic ganglion during development and after birth, but not in the auditory epithelium. Following noise damage, gicerin is transiently re-expressed on the damage receptor epithelium when active cell proliferation is observed in the epithelium. This positive reaction immediately disappears as immature short hair cells appear. These results suggest that gicerin may be associated with cell proliferation in the auditory epithelium, and play a role in neurite extension of the acoustic ganglion cells in conjunction with NOF.     

Coupling the cell cycle to development and regeneration of the inner ear

Cell cycle exit and acquirement of a postmitotic state is essential for the proper development of organs. In the present review, we examine the role of the cell cycle control in the sensory epithelia of the mammalian inner ear. We describe the roles of the core cell cycle regulators in the proliferation of prosensory cells and in the initiation and maintenance of terminal mitosis of the sensory epithelia. We also discuss how other intracellular signalling may influence the cell cycle. Finally, we address the question of whether manipulations of the cell cycle may have the potential to create replacement cells for the damaged inner sensory epithelia.     

Msx1 and Dlx5 function synergistically to regulate frontal bone development

The Msx and Dlx families of homeobox proteins are important regulators for embryogenesis. Loss of Msx1 in mice results in multiple developmental defects including craniofacial malformations. Although Dlx5 is widely expressed during embryonic development, targeted null mutation of Dlx5 mainly affects the development of craniofacial bones. Msx1 and Dlx5 show overlapping expression patterns during frontal bone development. To investigate the functional significance of Msx1/Dlx5 interaction in regulating frontal bone development, we generated Msx1 and Dlx5 double null mutant mice. In Msx1^?/?;Dlx5^?/? mice, the frontal bones defect was more severe than that of either Msx1^?/? or Dlx5^?/? mice. This aggravated frontal bone defect suggests that Msx1 and Dlx5 function synergistically to regulate osteogenesis. This synergistic effect of Msx1 and Dlx5 on the frontal bone represents a tissue specific mode of interaction of the Msx and Dlx genes. Furthermore, Dlx5 requires Msx1 for its expression in the context of frontal bone development. Our study shows that Msx1/Dlx5 interaction is crucial for osteogenic induction during frontal bone development. genesis 48:645–655, 2010. © 2010 Wiley‐Liss, Inc.