Notch signalling is needed to maintain, but not to initiate, the formation of prosensory patches in the chick inner ear

Notch signalling is well-known to mediate lateral inhibition in inner ear sensory patches, so as to generate a balanced mixture of sensory hair cells and supporting cells. Recently, however, we have found that ectopic Notch activity at an early stage can induce the formation of ectopic sensory patches. This suggests that Notch activity may have two different functions in normal ear development, acting first to promote the formation of the prosensory patches, and then later to regulate hair-cell production within the patches. The Notch ligand Serrate1 (Jag1 in mouse and humans) is expressed in the patches from an early stage and may provide Notch activation during the prosensory phase. Here, we test whether Notch signalling is actually required for prosensory patch development. When we block Notch activation in the chick embryo using the gamma-secretase inhibitor DAPT, we see a complete loss of prosensory epithelial cells in the anterior otocyst, where they are diverted into a neuroblast fate via failure of Delta1-dependent lateral inhibition. The cells of the posterior prosensory patch remain epithelial, but expression of Sox2 and Bmp4 is drastically reduced. Expression of Serrate1 here is initially almost normal, but subsequently regresses. The patches of sensory hair cells that eventually develop are few and small. We suggest that, in normal development, factors other than Notch activity initiate Serrate1 expression. Serrate1, by activating Notch, then drives the expression of Sox2 and Bmp4, as well as expression of the Serrate1 gene itself. The positive feedback maintains Notch activation and thereby preserves and perhaps extends the prosensory state, leading eventually to the development of normal sensory patches.     

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.     

Two contrasting roles for Notch activity in chick inner ear development: specification of prosensory patches and lateral inhibition of hair-cell differentiation

Lateral inhibition mediated by Notch is thought to generate the mosaic of hair cells and supporting cells in the inner ear, but the effects of the activated Notch protein itself have never been directly tested. We have explored the role of Notch signalling by transiently overexpressing activated Notch (NICD) in the chick otocyst. We saw two contrasting consequences, depending on the time and site of gene misexpression: (1) inhibition of hair-cell differentiation within a sensory patch; and (2) induction of ectopic sensory patches. We infer that Notch signalling has at least two functions during inner ear development. Initially, Notch activity can drive cells to adopt a prosensory character, defining future sensory patches. Subsequently, Notch signalling within each such patch mediates lateral inhibition, restricting the proportion of cells that differentiate as hair cells so as to generate the fine-grained mixture of hair cells and supporting cells.     

Jagged 1 regulates the restriction of Sox2 expression in the developing chicken inner ear: a mechanism for sensory organ specification

Hair cells of the inner ear sensory organs originate from progenitor cells located at specific domains of the otic vesicle: the prosensory patches. Notch signalling is necessary for sensory development and loss of function of the Notch ligand jagged 1 (Jag1, also known as serrate 1) results in impaired sensory organs. However, the underlying mechanism of Notch function is unknown. Our results show that in the chicken otic vesicle, the Sox2 expression domain initially contains the nascent patches of Jag1 expression but, later on, Sox2 is only maintained in the Jag1-positive domains. Ectopic human JAG1 (hJag1) is able to induce Sox2 expression and enlarged sensory organs. The competence to respond to hJag1, however, is confined to the regions that expressed Sox2 early in development, suggesting that hJag1 maintains Sox2 expression rather than inducing it de novo. The effect is non-cell-autonomous and requires Notch signalling. hJag1 activates Notch, induces Hes/Hey genes and endogenous Jag1 in a non-cell-autonomous manner, which is consistent with lateral induction. The effects of hJag1 are mimicked by Jag2 but not by Dl1. Sox2 is sufficient to activate the Atoh1 enhancer and to ectopically induce sensory cell fate outside neurosensory-competent domains. We suggest that the prosensory function of Jag1 resides in its ability to generate discrete domains of Notch activity that maintain Sox2 expression within restricted areas of an extended neurosensory-competent domain. This provides a mechanism to couple patterning and cell fate specification during the development of sensory organs.     

The Notch ligands DLL1 and JAG2 act synergistically to regulate hair cell development in the mammalian inner ear

The mammalian auditory sensory epithelium, the organ of Corti, contains sensory hair cells and nonsensory supporting cells arranged in a highly patterned mosaic. Notch-mediated lateral inhibition is the proposed mechanism for creating this sensory mosaic. Previous work has shown that mice lacking the Notch ligand JAG2 differentiate supernumerary hair cells in the cochlea, consistent with the lateral inhibitory model. However, it was not clear why only relatively modest increases in hair cell production were observed in Jag2 mutant mice. Here, we show that another Notch ligand, DLL1, functions synergistically with JAG2 in regulating hair cell differentiation in the cochlea. We also show by conditional inactivation that these ligands probably signal through the NOTCH1 receptor. Supernumerary hair cells in Dll1/Jag2 double mutants arise primarily through a switch in cell fate, rather than through excess proliferation. Although these results demonstrate an important role for Notch-mediated lateral inhibition during cochlear hair cell patterning, we also detected abnormally prolonged cellular proliferation that preferentially affected supporting cells in the organ of Corti. Our results demonstrate that the Notch pathway plays a dual role in regulating cellular differentiation and patterning in the cochlea, acting both through lateral inhibition and the control of cellular proliferation.     

Hesr1 and Hesr2 may act as early effectors of Notch signaling in the developing cochlea

In cochlear development, the Notch signaling pathway is required for both the early prosensory phase and a later lateral inhibition phase. While it is known that Hes genes are important downstream mediators of Notch function in lateral inhibition, it is not known what genes function as mediators of the early prosensory function of Notch. We report that two members of the Hes-related gene family, Hesr1 and Hesr2, are expressed in the developing cochlea at a time and place that makes them excellent candidates as downstream mediators of Notch during prosensory specification. We also show that treatment of cochlear explant cultures at the time of prosensory specification with a small-molecule inhibitor of the Notch pathway mimics the results of conditional Jag1 deletion. This treatment also reduces Hesr1 and Hesr2 expression by as much as 80%. These results support the hypothesis that Hesr1 and Hesr2 are the downstream mediators of the prosensory function of Notch in early cochlear development.     

Two Notch ligands, Dll1 and Jag1, are differently restricted in their range of action to control neurogenesis in the mammalian spinal cord

Background

Notch signalling regulates neuronal differentiation in the vertebrate nervous system. In addition to a widespread function in maintaining neural progenitors, Notch signalling has also been involved in specific neuronal fate decisions. These functions are likely mediated by distinct Notch ligands, which show restricted expression patterns in the developing nervous system. Two ligands, in particular, are expressed in non-overlapping complementary domains of the embryonic spinal cord, with Jag1 being restricted to the V1 and dI6 progenitor domains, while Dll1 is expressed in the remaining domains. However, the specific contribution of different ligands to regulate neurogenesis in vertebrate embryos is still poorly understood.

Methodology/Principal Findings

In this work, we investigated the role of Jag1 and Dll1 during spinal cord neurogenesis, using conditional knockout mice where the two genes are deleted in the neuroepithelium, singly or in combination. Our analysis showed that Jag1 deletion leads to a modest increase in V1 interneurons, while dI6 neurogenesis was unaltered. This mild Jag1 phenotype contrasts with the strong neurogenic phenotype detected in Dll1 mutants and led us to hypothesize that neighbouring Dll1-expressing cells signal to V1 and dI6 progenitors and restore neurogenesis in the absence of Jag1. Analysis of double Dll1;Jag1 mutant embryos revealed a stronger increase in V1-derived interneurons and overproduction of dI6 interneurons. In the presence of a functional Dll1 allele, V1 neurogenesis is restored to the levels detected in single Jag1 mutants, while dI6 neurogenesis returns to normal, thereby confirming that Dll1-mediated signalling compensates for Jag1 deletion in V1 and dI6 domains.

Conclusions/Significance

Our results reveal that Dll1 and Jag1 are functionally equivalent in controlling the rate of neurogenesis within their expression domains. However, Jag1 can only activate Notch signalling within the V1 and dI6 domains, whereas Dll1 can signal to neural progenitors both inside and outside its domains of expression.     

A mutation in the Lunatic fringe gene suppresses the effects of a Jagged2 mutation on inner hair cell development in the cochlea

Recent studies have demonstrated that the Notch signaling pathway regulates the differentiation of sensory hair cells in the vertebrate inner ear [1] [2] [3] [4] [5] [6] [7] [8] [9]. We have shown previously that in mice homozygous for a targeted null mutation of the Jagged2 (Jag2) gene, which encodes a Notch ligand, supernumerary hair cells differentiate in the cochlea of the inner ear [7]. Other components of the Notch pathway, including the Lunatic fringe (Lfng) gene, are also expressed during differentiation of the inner ear in mice [6] [7] [8] [9] [10]. In contrast to the Jag2 gene, which is expressed in hair cells, the Lfng gene is expressed in non-sensory supporting cells in the mouse cochlea [10]. Here we demonstrate that a mutation in the Lfng gene partially suppresses the effects of the Jag2 mutation on hair cell development. In mice homozygous for targeted mutations of both Jag2 and Lfng, the generation of supernumerary hair cells in the inner hair cell row is suppressed, while supernumerary hair cells in the outer hair cell rows are unaffected. We also demonstrate that supernumerary hair cells are generated in mice heterozygous for a Notch1 mutation. We suggest a model for the action of the Notch signaling pathway in regulating hair cell differentiation in the cochlear sensory epithelium.     

The Candidate Splicing Factor Sfswap Regulates Growth and Patterning of Inner Ear Sensory Organs

The Notch signaling pathway is thought to regulate multiple stages of inner ear development. Mutations in the Notch signaling pathway cause disruptions in the number and arrangement of hair cells and supporting cells in sensory regions of the ear. In this study we identify an insertional mutation in the mouse Sfswap gene, a putative splicing factor, that results in mice with vestibular and cochlear defects that are consistent with disrupted Notch signaling. Homozygous Sfswap mutants display hyperactivity and circling behavior consistent with vestibular defects, and significantly impaired hearing. The cochlea of newborn Sfswap mutant mice shows a significant reduction in outer hair cells and supporting cells and ectopic inner hair cells. This phenotype most closely resembles that seen in hypomorphic alleles of the Notch ligand Jagged1 (Jag1). We show that Jag1; Sfswap compound mutants have inner ear defects that are more severe than expected from simple additive effects of the single mutants, indicating a genetic interaction between Sfswap and Jag1. In addition, expression of genes involved in Notch signaling in the inner ear are reduced in Sfswap mutants. There is increased interest in how splicing affects inner ear development and function. Our work is one of the first studies to suggest that a putative splicing factor has specific effects on Notch signaling pathway members and inner ear development.     

Notch signaling regulates the pattern of auditory hair cell differentiation in mammals

The development of the mammalian cochlea is an example of patterning in the peripheral nervous system. Sensory hair cells and supporting cells in the cochlea differentiate via regional and cell fate specification. The Notch signaling components shows both distinct and overlapping expression patterns of Notch1 receptor and its ligands Jagged1 (Jag1) and Jagged2 (Jag2) in the developing auditory epithelium of the rat. On embryonic day 16 (E16), many precursor cells within the K?lliker's organ immunostained for the presence of both Notch1 and Jag1, while the area of hair cell precursors did not express either Notch1 and Jag1. During initial events of hair cell differentiation between E18 and birth, Notch1 and Jag1 expression predominated in supporting cells and Jag2 in nascent hair cells. Early after birth, Jag2 expression decreased in hair cells while the pattern of Notch1 expression now included both supporting cells and hair cells. We show that the normal pattern of hair cell differentiation is disrupted by alteration of Notch signaling. A decrease of either Notch1 or Jag1 expression by antisense oligonucleotides in cultures of the developing sensory epithelium resulted in an increase in the number of hair cells. Our data suggest that the Notch1 signaling pathway is involved in a complex interplay between the consequences of different ligand-Notch1 combinations during cochlear morphogenesis and the phases of hair cell differentiation.     

An Evolutionary-Conserved Function of Mammalian Notch Family Members as Cell Adhesion Molecules

Notch family members were first identified as cell adhesion molecules by cell aggregation assays in Drosophila studies. However, they are generally recognized as signaling molecules, and it was unclear if their adhesion function was restricted to Drosophila. We previously demonstrated that a mouse Notch ligand, Delta-like 1 (Dll1) functioned as a cell adhesion molecule. We here investigated whether this adhesion function was conserved in the diversified mammalian Notch ligands consisted of two families, Delta-like (Dll1, Dll3 and Dll4) and Jagged (Jag1 and Jag2). The forced expression of mouse Dll1, Dll4, Jag1, and Jag2, but not Dll3, on stromal cells induced the rapid and enhanced adhesion of cultured mast cells (MCs). This was attributed to the binding of Notch1 and Notch2 on MCs to each Notch ligand on the stromal cells themselves, and not the activation of Notch signaling. Notch receptor-ligand binding strongly supported the tethering of MCs to stromal cells, the first step of cell adhesion. However, the Jag2-mediated adhesion of MCs was weaker and unlike other ligands appeared to require additional factor(s) in addition to the receptor-ligand binding. Taken together, these results demonstrated that the function of cell adhesion was conserved in mammalian as well as Drosophila Notch family members. Since Notch receptor-ligand interaction plays important roles in a broad spectrum of biological processes ranging from embryogenesis to disorders, our finding will provide a new perspective on these issues from the aspect of cell adhesion.     

Notch ligand-dependent gene expression in human endometrial stromal cells

The purpose of this study is to investigate the expression patterns and role of Notch signaling in human endometrial cells. Notch receptors, Notch 1-3 were expressed in both endometrial epithelial and stromal cells. Notch ligands, Jag1 and Dll4 and Notch target genes, Hes1 and Hey1 were predominantly expressed in endometrial epithelial cells and scarce in stromal cells. Increased de novo synthesis of Dll4 or Jag1 in stromal cells by retroviral delivery significantly induced Hes1 and Hey1. Evaluations of global gene expression by microarrays revealed that more than 400 genes in stromal cells were significantly regulated by Jag1. Gene annotation-based functional analysis classified these genes into biological processes of cell adhesion, cell structure and motility, cell communication, cell cycle, and angiogenesis. This study provides evidence that Notch ligands control the Notch gene activities and may enhance development of human endometrium.     

Expression of Notch pathway genes in the embryonic mouse metanephros suggests a role in proximal tubule development

The interaction of neighboring cells via Notch signalling leads to cell fate determination, differentiation and patterning of highly organized tissues. Mice with targeted disruption of genes from the Notch signal transduction pathway display defects in the developing somites, neurogenic structures, blood vessels, heart and other organs. Recent studies have added requirements for Notch signalling during kidney, pancreas and thymus morphogenesis. Here, we describe the expression of all four receptors (Notch1-4), the five transmembrane ligands (Dll1, 3, 4, Jag1 and Jag2), intracellular effectors (the Hey genes) and extracellular modulators (Lfng, Mfng, Rfng) in the developing mouse metanephros. Our results point to a Lfng-dependent role for Notch signalling in the development of nephron segments, especially the proximal tubules.     

FGFR1-Frs2/3 Signalling Maintains Sensory Progenitors during Inner Ear Hair Cell Formation

Inner ear mechanosensory hair cells transduce sound and balance information. Auditory hair cells emerge from a Sox2-positive sensory patch in the inner ear epithelium, which is progressively restricted during development. This restriction depends on the action of signaling molecules. Fibroblast growth factor (FGF) signalling is important during sensory specification: attenuation of Fgfr1 disrupts cochlear hair cell formation; however, the underlying mechanisms remain unknown. Here we report that in the absence of FGFR1 signaling, the expression of Sox2 within the sensory patch is not maintained. Despite the down-regulation of the prosensory domain markers, p27^Kip1, Hey2, and Hes5, progenitors can still exit the cell cycle to form the zone of non-proliferating cells (ZNPC), however the number of cells that form sensory cells is reduced. Analysis of a mutant Fgfr1 allele, unable to bind to the adaptor protein, Frs2/3, indicates that Sox2 maintenance can be regulated by MAP kinase. We suggest that FGF signaling, through the activation of MAP kinase, is necessary for the maintenance of sensory progenitors and commits precursors to sensory cell differentiation in the mammalian cochlea.     

A cell autonomous role for the Notch ligand Delta-like 3 in αβ T-cell development

Notch signalling is critical to help direct T-cell lineage commitment in early T-cell progenitors and in the development of αβ T-cells. Epithelial and stromal cell populations in the thymus express the Notch DSL (Delta, Serrate and Lag2)ligands Delta-like 1 (Dll1), Delta-like 4 (Dll4), Jagged 1 and Jagged 2, and induce Notch signalling in thymocytes that express the Notch receptor. At present there is nothing known about the role of the Delta-like 3 (Dll3) ligand in the immune system. Here we describe a novel cell autonomous role for Dll3 in αβ T-cell development. We show that Dll3 cannot activate Notch when expressed in trans but like other Notch ligands it can inhibit Notch signalling when expressed in cis with the receptor. The loss of Dll3 leads to an increase in Hes5 expression in double positive thymocytes and their increased production of mature CD4(+) and CD8(+) T cells. Studies using competitive irradiation chimeras proved that Dll3 acts in a cell autonomous manner to regulate positive selection but not negative selection of autoreactive T cells. Our results indicate that Dll3 has a unique function during T-cell development that is distinct from the role played by the other DSL ligands of Notch and is in keeping with other recent studies indicating that Dll1 and Dll3 ligands have non-overlapping roles during embryonic development.     

MT1-MMP cleaves Dll1 to negatively regulate Notch signalling to maintain normal B-cell development

Notch signalling controls the differentiation of haematopoietic progenitor cells (HPCs). Here, we show that loss of membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP14), a cell surface protease expressed in bone marrow stromal cells (BMSCs), increases Notch signalling in HPCs and specifically impairs B-lymphocyte development. When co-cultured with BMSCs in vitro, HPCs differentiation towards B lymphocytes is significantly compromised on MT1-MMP-deficient BMSCs and this defect could be completely rescued by DAPT, a specific Notch signalling inhibitor. The defective B-lymphocyte development could also be largely rescued by DAPT in vivo. MT1-MMP interacts with Notch ligand Delta-like 1 (Dll1) and promotes its cleavage on cell surface in BMSCs. Ectopic MT1-MMP cleaves Dll1 and results in diminished Notch signalling in co-cultured cells. In addition, recombinant MT1-MMP cleaves a synthetic Dll1 peptide at the same site where MT1-MMP cleaves Dll1 on the cell surface. Our data suggest that MT1-MMP directly cleaves Dll1 on BMSCs to negatively regulate Notch signalling to specifically maintain normal B-cell development in bone marrow.