Notch
Notch and ear development The sensory patches in the vertebrate inner ear are similar
in function to the mechanosensory bristles of a fly, and
consist of a similar set of cell types. If they are truly
homologous structures, they should also develop by similar
mechanisms. The genesis of the neurons, hair
cells and supporting cells that form the sensory patches in
the inner ear of the chick was examined. These cells all arise from the otic
epithelium, and are produced normally even in otic
epithelium cultured in isolation, confirming that their
production is governed by mechanisms intrinsic to the
epithelium. The otic placode is first visible at 1.5 days of incubation (E1.5)
as a thickening in the head ectoderm next to the hindbrain at the
level of rhombomeres 5 and 6. The placode invaginates to form
a cup, which closes and pinches off from the head ectoderm to
become a pear-shaped otocyst. Over the following four or five
days complex shape changes take place, converting the otocyst
into a membranous labyrinth with dorsal semicircular canals and utricle
and ventral saccule and banana-shaped cochlea.
Neurogenesis occurs at the beginning of this period (E2-E3.5);
hair-cell differentiation towards its end (from E5 onwards).
The neuronal sublineage becomes
separate from the epithelial: between E2 and E3.5,
neuroblasts delaminate from the otocyst. The neuroblasts
then give rise to a mixture of neurons and neuroblasts,
while the sensory epithelial cells diversify to form a mixture
of hair cells and supporting cells. The epithelial patches
where this occurs are marked from an early stage by
uniform and maintained expression of the Notch ligand
Serrate1. The Notch ligand Delta1 (Dl1) is also expressed, but
transiently and in scattered cells: it is seen both early,
during neuroblast segregation, where it appears to be in the
nascent neuroblasts, and again later, in the ganglion and in
differentiating sensory patches, where it appears to be in
the nascent hair cells, disappearing as they mature. Delta-Notch-mediated lateral inhibition may thus act at each
developmental branchpoint to drive neighboring cells
along different developmental pathways. These findings
indicate that the sensory patches of the vertebrate inner ear
and the sensory bristles of a fly are generated by minor
variations of the same basic developmental program, in
which cell diversification driven by Delta-Notch and/or
Serrate-Notch signaling plays a central part (Adam, 1998).
By early stage 11 (40 hours), expression of Notch1 marks
out an ectodermal patch that includes the whole otic placode;
within the patch, expression is uniform. By the end
of stage 11 (about 3 hours later), a few scattered cells
expressing Dl1 begin to be seen in the anterior part of the
placode. The number of cells expressing Dl1 increases rapidly,
but these remain confined to the anterior half of the placode
(becoming anteroventral in the cup) and continue as only a
scattered subset of the cell population in that region.
To follow neurogenesis and to see how it relates to Dl1
expression, four antibodies were used: Islet1/2, TuJ1 and
BEN (each of which detect neuronal antigens whose expressions are reported to begin early in neuronal differentiation, before
neurofilaments are seen in the cell), and 3A10, which binds to a
neurofilament-associated epitope.
In the otic epithelium, immunostaining with
Islet1/2, TuJ1 and BEN is first seen at stage 12/13 (48-49 hours), 6-7
hours after Dl1 expression begins. By stage 14/15 (52
hours), staining with Islet1/2, TuJ1 and BEN is
concentrated in the anteroventral part of the otic cup. Islet1/2,
as a nuclear marker, gives the most precise indication of the
behavior of individual cells: within the anteroventral domain,
the cells expressing Islet1/2 antigen form a scattered subset of
the epithelial population. The cells expressing
Islet1/2 and TuJ1 are concentrated basally in the epithelium,
and some can be seen straddling the basal lamina, as though in
the act of delaminating.
Doubly stained sections show that the Islet1/2 domain
coincides with the Dl1 expression domain, but that the
individual cells in this region never express both markers
simultaneously. By analogy with the embryonic
central nervous system, this suggests that the Dl1-expressing
cells in the ear epithelium are neuronal precursors, expressing
Dl1 transiently before switching on expression of markers of
neuronal differentiation.
At stage 14-15 (52 hours), a few cells are seen
expressing Islet1/2, TuJ1 and BEN antigens that have escaped
to form the first rudiment of the cochleovestibular ganglion,
pressed close against the anteroventral otic epithelium. On the
basis of these observations, the anteroventral
patch of expression of Dl1, Islet1/2, TuJ1 and BEN in the otic
epithelium has been identified as the site of neurogenesis. A similar pattern of
labelling persists in this neurogenic patch for about 36 hours,
up to stage 21-22 (84-90 hours). Throughout this period
there is a continuing exodus of cells expressing Islet1/2, TuJ1
and BEN from the otic epithelium into the developing ganglion (Adam, 1998).
The delaminating cells are neuroblasts rather than
postmitotic neurons. 3A10 staining is not seen in the ear until at least stage 17 (58
hours), 6 hours after delamination has begun, and it is
confined to the ganglion. The 3A10-positive cells are
identifiable as young bipolar neurons, with axons and dendrites. Over the subsequent days, the number of these neurons in the ganglion steadily increases.
From previous [3H]thymidine studies of cell division in the
ganglion, it is clear that the ganglion
precursor cells delaminating from the otic epithelium are not
postmitotic nascent neurons, but neuroblasts capable of dividing
before they differentiate. The following three
observations confirm this: (1) Islet-positive cells can be seen in
mitosis both in the neurogenic patch in the otic epithelium and in the developing cochleovestibular ganglion,
and this matches findings with BrdU labelling.
(2) BEN expression persists in the target epithelium as
well as in the developing neurons.
(3) The Islet1/2 and TuJ1 antigens continue to be expressed in the
ganglion, but in the otic epithelium cells expressing antigens, such expression has
disappeared by stage 21-22 (84-90 hours), corresponding
to the end of delamination of neuronal precursors from the
otocyst. BEN, however, behaves differently. In the ganglion, its
expression is similar to that of Islet1/2 and TuJ1, but in the otic
epithelium it persists after Islet1/2 and TuJ1 have disappeared,
and marks the region that is invaded by processes from the
ganglion cells, i.e. the presumptive sensory area.
Thus BEN labels both the neurons and their peripheral target
epithelium. Homophilic interactions mediated by BEN (a cell-surface
adhesion molecule) may help the dendrites of the
cochleovestibular neurons, as they grow back into the otic
epithelium, to recognise the sensory patch in which they must
make synapses (Adam, 1998).
When comparing the development of sensory bristles in Drosophila and sensory patches
in the vertebrate ear, it has been concluded that both tissue types are generated by essentially the same
developmental program.
Each insect sensory bristle is a functional unit formed from the
progeny of a sensory mother cell (SMC): this cell is singled out
from a proneural cluster in the insect epidermis, and (in the case
of a standard mechanosensory bristle) divides twice to generate
four different cells. At each division of the SMC and its progeny,
Delta and Serrate, act together in a quasi-redundant fashion
as ligands for Notch, mediating lateral inhibition to force the sister
cells to adopt different fates. In the first division, one daughter (the
daughter delivering lateral inhibition) becomes committed as a
neuroblast while the other becomes committed as a sensory
epithelial precursor. In the second division, the daughters of the
neuroblast become, respectively, a neuron (delivering lateral
inhibition) and a neural sheath cell, while the daughters of the
sensory epithelial precursor become, respectively, a bristle shaft
cell (delivering lateral inhibition) and a bristle socket cell. Some
SMCs follow variants of this program: in chemosensory bristles,
for example, the neuroblast divides several times to generate 3-
5 neurons; in non-innervated bristles
of the posterior wing margin, conversely, neurons are missing (Adam, 1998).
The sensory neuron of the Drosophila bristle corresponds to the sensory
neuron of the ear; the shaft cell, presumably, to the hair cell of
the ear; and the socket cell to the supporting
cell. The Drosophila neural sheath cell has no such obvious counterpart: the
glial cells in the cochleovestibular ganglion derive from the
neural crest, not the otic epithelium. One might, however, compare the neural sheath
cell of the bristle to a second-generation neuroblast in the
cochleovestibular ganglion -- both of them are daughters of first-generation
neuroblasts but have not differentiated into neurons.
Assuming these correspondences between the cell types, the
correspondences in the developmental program can be inferred
directly. At the outset, however, there is a contrast.
Whereas each bristle is typically isolated from the next by
intervening epidermis, each sensory patch in the ear consists of
a mass of contiguous hair cells and supporting cells, with no non-sensory
cells between them. Thus it seems that the counterpart
of the SMC is not a single isolated cell, but a cluster of contiguous
sensory precursor cells (SPCs) that coexist, instead of competing
by lateral inhibition. (In Drosophila, SMCs likewise develop in
contiguity, exceptionally, at the wing margin). If the ear/bristle parallel is drawn in this way,
it is possible to relate all the subsequent steps of sensory patch
development to those of bristle development, with Delta/Serrate-Notch
signaling acting repeatedly in a similar way in the two
systems. The singling-out of neuroblasts in the ear corresponds
to the determination of one of the two daughters of an SMC as a
neuroblast. The production of neurons from neuroblasts in the
cochleovestibular ganglion corresponds to the production of a
neuron or of several neurons from the bristle neuroblast. And the
genesis of hair cells and supporting cells from otic sensory
epithelial precursors corresponds to the genesis of bristle shaft
cells and supporting cells from sensory epithelial precursors in
the insect epidermis. The two systems differ, it is true, in the
numbers of cell divisions that occur at each step; but this is
variable even between types of bristles in the fly.
In addition to the above systematic parallels in the
developmental program, there are other facts that suggest a
conserved process. For example, Pax2, along with its close
relatives Pax5 and Pax8, is strongly expressed in the early ear
rudiment and is required for development
of the cochlea; and its homolog in
Drosophila, Sparkling, is strongly expressed in the precursor
cells of the sensory bristles.
It seems clear that the mechanosensory organs of flies and
vertebrates are fundamentally similar, not only in function and
architecture, but also in the developmental programs that
generate their precisely patterned arrays of cell types. The
precise correspondence identified here should help in
the search for other molecules that have a conserved role in the
two systems (Adam, 1998).
The cochlea and vestibular structures of the inner ear labyrinth develop from the otic capsule via step-wise regional and cell fate
specification. Each inner ear structure contains a sensory epithelium, composed of hair cells, the mechanosensory transducers,
and supporting cells. The spatio-temporal expression of genes in the Notch signaling pathway, Notch receptors
(Notch1-4) and two ligands, Jagged1 and Delta1, were examined in the developing mammalian inner ear. Notch1 and
Jagged1 are first expressed in the otic vesicle, likely involved in differentiation of the VIIIth nerve ganglion neurons, and
subsequently within the inner ear sensory epithelia, temporally coincident with initial hair cell differentiation. Notch1
expression is specific to hair cells and Jagged1 to supporting cells. Their expression persists into adult. Notch2, Notch3,
Notch4, and Delta1 are excluded from the inner ear epithelia. These data support the hypothesis that Notch signaling is
involved in hair cell differentiation during inner ear morphogenesis (Lewis, 1998).
Mechanosensory hair cells in the sensory patches of the vertebrate ear are interspersed among supporting cells,
forming a fine-grained pattern of alternating cell types. Analogies with Drosophila mechanosensory bristle
development suggest that this pattern could be generated through lateral inhibition mediated by Notch signaling. In the
zebrafish ear rudiment, homologs of Notch are widely expressed, while the Delta homologs deltaA, deltaB and
deltaD, coding for Notch ligands, are expressed in small numbers of cells in regions where hair cells are soon to
differentiate. This suggests that the delta-expressing cells are nascent hair cells, in agreement with findings for
Delta1 in the chick. According to the lateral inhibition hypothesis, the nascent hair cells, by expressing Delta protein,
would inhibit their neighbours from becoming hair cells, forcing them to be supporting cells instead. The zebrafish
mind bomb mutant has abnormalities in the central nervous system, somites, and elsewhere, diagnostic of a failure of
Delta-Notch signaling: in the CNS, it shows a neurogenic phenotype accompanied by misregulated delta gene
expression. Similar misregulation of delta genes is seen in the ear, along with misregulation of a Serrate homolog,
serrateB, coding for an alternative Notch ligand. Most dramatically, the sensory patches in the mind bomb ear consist
solely of hair cells, which are produced in great excess and prematurely; at 36 hours post fertilization, there are more
than ten times as many as normal, while supporting cells are absent. A twofold increase is seen in the number of otic
neurons also. The findings are strong evidence that lateral inhibition mediated by Delta-Notch signaling controls the
pattern of sensory cell differentiation in the ear. Although the molecular nature of the mib gene remains to be discovered, the mutant provides a way to test the role of the Notch signaling pathway in the various tissues of the cell (Haddon, 1998).
The mammalian cochlea contains an invariant mosaic of sensory hair cells and non-sensory supporting
cells reminiscent of invertebrate structures such as the compound eye in Drosophila melanogaster. The
sensory epithelium in the mammalian cochlea (the organ of Corti) contains four rows of
mechanosensory hair cells: a single row of inner hair cells and three rows of outer hair cells. Each hair
cell is separated from the next by an interceding supporting cell, forming an invariant and alternating
mosaic that extends the length of the cochlear duct. Previous results suggest that determination of cell
fates in the cochlear mosaic occurs via inhibitory interactions between adjacent progenitor cells (lateral
inhibition). Cells populating the cochlear epithelium appear to constitute a developmental equivalence
group in which developing hair cells suppress differentiation in their immediate neighbors through
lateral inhibition. These interactions may be mediated through the Notch signaling pathway, a
molecular mechanism that is involved in the determination of a variety of cell fates. Genes encoding the receptor protein Notch1 and its ligand, Jagged 2, are expressed in alternating cell
types in the developing sensory epithelium. In addition, genetic deletion of Jag2 results in a significant
increase in sensory hair cells, presumably as a result of a decrease in Notch activation. These results
provide direct evidence for Notch-mediated lateral inhibition in a mammalian system and support a role
for Notch in the development of the cochlear mosaic (Lanford, 1999).
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 show 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 Kolliker's organ
immunostain for the presence of both Notch1 and Jag1,
while the area of hair cell precursors express
neither Notch1 nor Jag1. During initial events of hair cell
differentiation between E18 and birth, Notch1 and Jag1
expression predominates in supporting cells and Jag2 in
nascent hair cells. Early after birth, Jag2 expression
decreases in hair cells while the pattern of Notch1
expression now includes both supporting cells and hair
cells. 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 results in an increase in the number of
hair cells. These 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 (Zine, 2000).
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. The role of Notch signalling was explored by transiently overexpressing activated Notch (NICD) in the chick otocyst. Two contrasting consequences ensued, 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. It is inferred 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 (Daudet, 2005).
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, 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. This study tested whether Notch signalling is actually required for prosensory patch development. When Notch activation was blocked in the chick embryo using the gamma-secretase inhibitor DAPT, a complete loss of prosensory epithelial cells was seen 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. It is suggested 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 (Daudet, 2007).
Otic neuronal precursors are the first cells to be specified and do so in the anterior domain of the otic placode, the proneural domain. The present study explored the early events of otic proneural regionalization in relation to the activity of the Notch signaling pathway. The proneural domain was characterized by the expression of Sox3, Fgf10 and members of the Notch pathway such as Delta1, Hes5 and Lunatic Fringe. The complementary non-neural domain expressed two patterning genes, Lmx1b and Iroquois1, and the members of the Notch pathway, Serrate1 and Hairy1. Fate map studies and double injections with DiI/DiO showed that labeled cells remained confined to anterior or posterior territories with limited cell intermingling. To explore whether Notch signaling pathway plays a role in the initial regionalization of the otic placode, Notch activity was blocked by a γ-secretase inhibitor (DAPT). Notch blockade induced the expansion of non-neural genes, Lmx1 and Iroquois1, into the proneural domain. Combined gene expression and DiI experiments showed that these effects were not due to migration of non-neural cells into the proneural domain, suggesting that Notch activity regulates the expression of non-neural genes. This was further confirmed by the electroporation of a dominant-negative form of the Mastermind-like1 gene that caused the up-regulation of Lmx1 within the proneural domain. In addition, Notch pathway was involved in neuronal precursor selection, probably by a classical mechanism of lateral inhibition. It is proposed that the regionalization of the otic domain into a proneural and a non-neural territory is a very early event in otic development, and that Notch signaling activity is required to exclude the expression of non-neural genes from the proneural territory (Abelló, 2007).
Notch and tooth development Recent data suggest that dental cells utilize the evolutonarily conserved Notch-mediated intercellular
signaling pathway to regulate their fates. The expression and regulation of Delta1, a
transmembrane ligand of the Notch receptors, during mouse odontogenesis is described. Delta1 is weakly
expressed in dental epithelium during tooth initiation and morphogenesis, but during cytodifferentiation,
expression is upregulated in the epithelium-derived ameloblasts and the mesenchyme-derived
odontoblasts. The expression pattern of Delta1 in ameloblasts and odontoblasts is complementary to
Notch1, Notch2, and Notch3 expression in adjacent epithelial and mesenchymal cells. Notch1 and
Notch2 are upregulated in explants of dental mesenchyme adjacent to implanted cells expressing
Delta1, suggesting that feedback regulation by Delta-Notch signaling ensures the spatial segregation of
Notch receptors and ligands. TGFbeta1 and BMPs induce Delta1 expression in dental mesenchyme
explants at the stage at which Delta1 is upregulated in vivo, but not at earlier stages. In contrast to the
Notch family receptors and their ligand Jagged1, expression of Delta1 in the tooth germ is not affected
by epithelial-mesenchymal interactions, showing that the Notch receptors and their two ligands Jagged1
and Delta1 are subject to different regulations (Matsiadis, 1998).
Notch and eye development Genes that can direct the formation of glia
in the retina have been identified. rax, a homeobox gene (Drosophila homolog Rx); Hes1, a basic helix-loop-helix gene, and notch1, a transmembrane receptor gene, are all expressed in retinal progenitor cells,
downregulated in differentiated neurons, and expressed in Müller glia. Retroviral transduction of any of these genes results in expression of glial markers. In contrast,
misexpression of a dominant-negative Hes1 gene reduces the number of glia. Cotransfection of rax with reporter constructs containing the Hes1 or notch1
regulatory regions leads to the upregulation of reporter transcription. These data suggest a regulatory heirarchy that controls the formation of glia at the expense of
neurons (Furukawa, 2000).
Thus, rax, Hes1, and notch1 are expressed by retinal progenitor cells and by differentiating Müller glia. In addition, when individually transduced, all three genes are capable of promoting the formation of cells that express markers of Müller glia. Since all three of these genes are presumably transcription factors, these observations raise the possibility that they either directly or indirectly regulate each other. There is evidence that activated notch1 directly upregulates Hes1. Evidence is provided that rax leads to upregulation of Hes1 and notch1. Following infection with rax-GFP virus, both notch1 and Hes1 RNA are detected using a RT/PCR assay. The upregulation of Hes1 and notch1 by rax may be direct. Reporter constructs with either the notch1 or Hes1 regulatory regions showed a 5-fold induction in RNA levels when rax is cotransfected. The Hes1 upstream region encodes two putative sites for a paired-type homeobox gene, such as rax. The sequence of the 11 kb notch1 regulatory regions is not yet known, but the data predict that such a site is present in notch1 as well. Since rax is expressed prior to notch1 or Hes1 in the retinal anlagen, it is likely that at least the initial period of rax transcription is independent of Hes1 and notch, while the subsequent expression of Hes1 and notch1 may be dependent upon rax (Furukawa, 2000).
The Gal4-UAS technique has been used to misexpress a
constitutively active Notch receptor variant (notch1a-intra)
in the developing zebrafish retina. This is the first study to
use this technique to misexpress genes and assess their
function in neural development of the zebrafish. Expression
of activated Notch1a either ubiquitously, driven by a heat-shock70
promoter, or in a spatially regulated manner,
controlled by the deltaD promoter, causes a block in
neuronal differentiation that affects all cell types.
Developing cells take on either a glial fate or remain
undifferentiated. A large number of cells eventually
undergo apoptosis. These phenotypic effects of activated
Notch1a are expressed cell autonomously. Cells within
central regions of the retina adopt a glial fate if they express
activated Notch1a in a time window that extends from 27
to 48 hours postfertilization. This period corresponds
mainly to the time of origin of ganglion cells in the normal
retina. Activation of Notch1a at later stages results in
defects in cell type specification that remain restricted to
the ciliary marginal zone, whereas neuronal types are
specified normally within the central region. These
observations indicate that glial differentiation is initiated
by Notch1a-intra expressing cells, which become
postmitotic in the same time window. These results strongly
suggest that Notch1a instructs a certain cell population to
enter gliogenesis, and keeps the remaining cells in an
undifferentiated state. Some or all of these cells will
eventually succumb to apoptosis. These results represent
the first in vivo evidence that Notch signaling may perform
such an instructive function in retinal development in the
zebrafish (Scheer, 2001).
Loss of Pax 6 function leads to an eyeless phenotype in both mammals and insects, and ectopic expression of both the Drosophila and
the mouse gene leads to the induction of ectopic eyes in Drosophila, which suggests that Pax 6 might be a universal master
control gene for eye morphogenesis. This study reports the reciprocal experiment in which the RNAs of the Drosophila Pax 6 homologs,
eyeless and twin of eyeless, are transferred into a vertebrate embryo; i.e., early Xenopus embryos at the 2- and 16-cell stages. In both
cases, ectopic eye structures are formed. To understand the genetic program specifying eye morphogenesis, the
regulatory mechanisms of Pax 6 expression that initiates eye development have been examined. Notch signaling regulates the expression of eyeless and twin of eyeless in Drosophila. In Xenopus, activation of Notch signaling also induces eye-related gene expression, including Pax 6, in isolated animal caps. In Xenopus embryos, the activation of Notch signaling causes eye duplications and proximal eye defects, which are also induced by overexpression of eyeless and twin of eyeless. These findings indicate that the gene regulatory cascade is similar in vertebrates and invertebrates (Onuma, 2002).
Notch-Delta signaling has been implicated in several alternative modes of function in the vertebrate retina. To further investigate these functions, retinas were examined from zebrafish embryos in which bidirectional Notch-Delta signaling was inactivated either by the mind bomb (mib) mutation, which disrupts E3 ubiquitin ligase activity, or by treatment with gamma-secretase inhibitors, which prevent intramembrane proteolysis of Notch and Delta. Inactivating Notch-Delta signaling does not prevent differentiation of retinal neurons, but it does disrupt spatial patterning in both the apical-basal and planar dimensions of the retinal epithelium. Retinal neurons differentiate, but their laminar arrangement is disrupted. Photoreceptor differentiation is initiated normally, but its progression is slowed. Although confined to the apical retinal surface as in normal retinas, the planar organization of cone photoreceptors is disrupted: cones of the same spectral subtype are clumped rather than regularly spaced. In contrast to neurons, Müller glia fail to differentiate suggesting an instructive role for Notch-Delta signaling in gliogenesis (Bernardos, 2005).
The principal finding of this study was that retinal neurons differentiate when Notch-Delta signaling is disrupted, but Müller glia do not. While the possibility that Müller glia are produced but fail to differentiate cannot be excluded, no evidence was found of expression of early markers of Müller glia, such as glutamine synthetase, GFAP or her6/hes1 (an ortholog of Hes1 in mammals), suggesting that retinal gliogenesis was blocked. In contrast, in both mib−/− and γ-secretase-inhibited embryos, markers of neuronal differentiation were expressed in the retina at the appropriate developmental stages, although normal laminar organization of the inner retina was disrupted. Most retinal ganglion cells were positioned near the inner retinal surface, but they were not organized into a continuous, discrete layer. The inner nuclear layer was similarly disrupted. In contrast, stratification of the outer nuclear layer was approximately normal, and a single row of differentiating photoreceptors with columnar nuclei formed along the apical retinal surface. However, the photoreceptors were not appropriately positioned in the planar dimension. In the normal zebrafish retina, photoreceptors are arranged in a highly organized, planar mosaic pattern with precise spatial relationships among the four different cone photoreceptors (red, green, blue, and ultraviolet) and the rod photoreceptors. This regular arrangement and the progression of photoreceptor differentiation, which normally proceeds in a wave across the retina, were disrupted in mib−/− mutants and γ-secretase-treated embryos. Although the cellular and molecular mechanisms that pattern the photoreceptor mosaic in the teleost retina are unknown, Notch-Delta signaling is important for photoreceptor cell identity and spacing in the compound eye of Drosophila, and the current results suggest that it may play a similar role in planar patterning of photoreceptors in zebrafish (Bernardos, 2005).
During the development of the central nervous system, cell proliferation and
differentiation are precisely regulated. In the vertebrate eye, progenitor
cells located in the marginal-most region of the neural retina continue to
proliferate for a much longer period compared to the ones in the central
retina, thus showing stem-cell-like properties. Wnt2b is expressed in the
anterior rim of the optic vesicles, and has been shown to control
differentiation of the progenitor cells in the marginal retina. Stable overexpression of Wnt2b in retinal explants inhibits cellular differentiation and induces continuous growth of the tissue. Notably, Wnt2b maintained the undifferentiated progenitor cells in the explants even under the conditions where Notch signaling is blocked. Wnt2b downregulates the expression of multiple proneural bHLH genes as well as Notch. In addition, expression of Cath5 under the control of an exogenous promoter suppresses the negative effect of Wnt2b on neuronal differentiation. Importantly, Wnt2b inhibits neuronal differentiation independently of cell cycle progression. It is proposed that Wnt2b maintains the naive state of marginal progenitor cells by attenuating the expression of both proneural and neurogenic genes, thus preventing those cells from launching out into the differentiation cascade regulated by proneural genes and Notch (Kudo, 2005).
Integrity and preservation of a transparent cornea are essential for good vision. The corneal epithelium is stratified and nonkeratinized and is maintained and repaired by corneal stem cells. Notch1 signaling is essential for cell fate maintenance of corneal epithelium during repair. Inducible ablation of Notch1 in the cornea combined with mechanical wounding show that Notch1-deficient corneal progenitor cells differentiate into a hyperplastic, keratinized, skin-like epithelium. This cell fate switch leads to corneal blindness and involves cell nonautonomous processes, characterized by secretion of fibroblast growth factor-2 (FGF-2) through Notch1-/- epithelium followed by vascularization and remodeling of the underlying stroma. Vitamin A deficiency is known to induce a similar corneal defect in humans (severe xerophthalmia). Accordingly, it was found that Notch1 signaling is linked to vitamin A metabolism by regulating the expression of cellular retinol binding protein 1 (CRBP1), required to generate a pool of intracellular retinol (Vauclair, 2007).
Notch and lung development Factors controlling the differentiation of the multipotent embryonic lung endoderm and mesoderm are poorly understood. Recent evidence
that Delta-like 1 (Dll1) and other genes in the Notch/Delta signaling pathway are expressed in the embryonic mouse lung suggests that this
pathway is important for cell fate decisions and/or the differentiation of lung cell types. The localization of transcripts of
several genes encoding members of the Notch/Delta pathway in the early mouse lung is reported. Most genes are expressed in specific populations and
so may contribute to cell diversification (Post, 2000).
Notch1 expression is seen in the distal endoderm at all
times examined (E11.5-E13.5). By contrast,
Notch2 and Notch3 are expressed throughout the mesenchyme, although Notch3 transcripts are also found at low
levels in the endoderm. Notch4 expression
is confined to the endothelium of the blood vessels.
Dll1 is expressed within the
respiratory epithelium after E13.5. Dll3 expression
was not observed in the developing mouse lung at any age
examined. Jagged1 (Jag1) transcripts are
present in the mesenchyme and blood vessels,
while Jag2 expression is observed in the peripheral
mesenchyme underlying the surface mesothelium.
Radical and Lunatic fringe proteins are believed to modulate the binding of ligand to the Notch receptors. Lfng
RNA is localized to the developing endoderm of both the
trachea and respiratory tree while Rfng expression
is ubiquitous throughout the endoderm and mesenchyme (Post, 2000).
The localized expression of Dll1 during early lung development was studied in more detail using lungs from Dll1LacZ
heterozygous animals. Two distinct positive cell populations were found.
(1) LacZ-positive cells are observed in isolated clusters
in the secondary bronchi after E13.5. As gestation
progresses, positive cells increase in number and can be
found within more terminal branches of the bronchioles,
often at branch points.
Postnatally, fewer positive endodermal cells are found,
only within the deepest parts of the lung tissue; this pattern
is maintained in the adult. The early expression
pattern of Dll1 and the similar temporal-spatial pattern of
expression seen for Mash1, encoding a mediator of Dll1
function, suggests that the Dll1-positive cells are neuroendocrine cells (NE cells). Mash1 null
mice are viable but lack NE cells in the adult lung. (2) A second pattern of Dll1 expression was seen
after postnatal day 7 throughout the lung tissue.
Upon sectioning, positive cells were identified as endothelial cells lining the lung vasculature (Post, 2000).
Mice carrying loss-of-function mutations in certain Notch pathway genes display increased and accelerated pancreatic endocrine development, leading to depletion of precursor cells followed by pancreatic hypoplasia. Investigated here was the effect of expressing a constitutively active form of the Notch1 receptor (Notch1ICD) in the developing pancreas using the pdx1 promoter. At e10.5 to e12.5, a disorganized pancreatic epithelium was observed with reduced numbers of endocrine cells, confirming a repressive activity of Notch1 upon the early differentiation program. Subsequent branching morphogenesis is impaired and the pancreatic epithelium forms cyst-like structures with ductal phenotype containing a few endocrine cells but completely devoid of acinar cells. The endocrine cells that do form show abnormal expression of cell type-specific markers. These observations show that sustained Notch1 signaling not only significantly represses endocrine development, but also fully prevents pancreatic exocrine development, suggesting that a possible role of Notch1 is to maintain the undifferentiated state of common pancreatic precursor cells (Hald, 2003).
Notch signaling regulates cell fate decisions in a variety of adult and embryonic tissues, and represents a characteristic feature of exocrine pancreatic cancer. In developing mouse pancreas, targeted inactivation of
Notch pathway components has defined a role for Notch in regulating early endocrine differentiation, but has been less informative with respect to a possible role for Notch in regulating subsequent exocrine differentiation events. Activated Notch and Notch target genes actively repress completion of an acinar cell differentiation program in developing mouse and zebrafish pancreas. In developing mouse pancreas, the Notch target gene Hes1 is co-expressed with Ptf1-P48 (a bHLH transcription factor) in exocrine precursor cells, but not in differentiated amylase-positive acinar cells. Using lentiviral delivery systems to induce ectopic Notch pathway activation in explant cultures of E10.5 mouse dorsal pancreatic buds, it has been found that both Hes1 and Notch1-IC repress acinar cell differentiation, but not Ptf1-P48 expression, in a cell-autonomous manner. Ectopic Notch activation also delays acinar cell differentiation in developing zebrafish pancreas. Further evidence of a role for endogenous Notch in regulating exocrine pancreatic differentiation was provided by examination of zebrafish embryos with homozygous mindbomb mutations, in which Notch signaling is disrupted. mindbomb-deficient embryos display accelerated
differentiation of exocrine pancreas relative to wild-type clutchmate controls. A similar phenotype was induced by expression of a dominant-negative
Suppressor of Hairless [Su(H)] construct, confirming that Notch actively represses acinar cell differentiation during zebrafish pancreatic development. Using transient transfection assays involving a
Ptf1-responsive reporter gene, it was further demonstrated that Notch and
Notch/Su(H) target genes directly inhibit Ptf1 activity, independent of
changes in expression of Ptf1 component proteins. These results define a
normal inhibitory role for Notch in the regulation of exocrine pancreatic
differentiation (Esni, 2004).
Relatively little is known about the developmental signals that specify the types and numbers of pancreatic cells. Previous studies suggested that Notch signaling in the pancreas inhibits differentiation and promotes the maintenance of progenitor cells, but it remains unclear whether Notch also controls cell fate choices as it does in other tissues. To study the impact of Notch in progenitors of the β cell lineage, mice were generated that express Cre-recombinase under control of the Pax4 promoter. Lineage analysis of Pax4+ cells demonstrates they are specified endocrine progenitors that contribute equally to four islet cell fates, contrary to expectations raised by the dispensable role of Pax4 in the specification of the α and PP subtypes. In addition, activation of Notch in Pax4+ progenitors inhibits their differentiation into α and β endocrine cells and shunts them instead toward a duct fate. These observations reveal an unappreciated degree of developmental plasticity among early endocrine progenitors and raise the possibility that a bipotent duct-endocrine progenitor exists during development. Furthermore, the redirection of Pax4+ cells from α and β endocrine fates toward a duct cell type suggests a positive role for Notch signaling in duct specification and is consistent with the more widely defined role for Notch in cell fate determination (Greenwood, 2007).
Canonical Notch signaling is thought to control the endocrine/exocrine decision in early pancreatic progenitors. Later, RBP-Jkappa interacts with Ptf1a and E12 to promote acinar differentiation. To examine the involvement of Notch signaling in selecting specific endocrine lineages, this pathway was deregulated by targeted deletion of presenilin1 and presenilin2, the catalytic core of gamma-secretase, in Ngn3- or Pax6-expressing endocrine progenitors. Surprisingly, whereas Pax6(+) progenitors were irreversibly committed to the endocrine fate, it was discovered that Ngn3(+) progenitors were bipotential in vivo and in vitro. When presenilin amounts are limiting, Ngn3(+) progenitors default to an acinar fate; subsequently, they expand rapidly to form the bulk of the exocrine pancreas. gamma-Secretase inhibitors confirmed that enzymatic activity was required to block acinar fate selection by Ngn3 progenitors. Genetic interactions identified Notch2 as the substrate, and suggest that gamma-secretase and Notch2 act in a noncanonical titration mechanism to sequester RBP-Jkappa away from Ptf1a, thus securing selection of the endocrine fate by Ngn3 progenitors. These results revise the current view of pancreatic cell fate hierarchy, establish that Ngn3 is not in itself sufficient to commit cells to the endocrine fate in the presence of Ptf1a, reveal a noncanonical action for Notch2 protein in endocrine cell fate selection, and demonstrate that acquisition of an endocrine fate by Ngn3(+) progenitors is gamma-secretase-dependent until Pax6 expression begins (Cras-Méneur, 2009).
The Notch pathway regulates cell fate determination in numerous developmental processes. Notch2 acts non-redundantly to control the processes of nephron segmentation through an Rbp-J-dependent process. Notch1 and Notch2 are detected in the early renal vesicle. Genetic analysis reveals that only Notch2 is required for the differentiation of proximal nephron structures (podocytes and proximal convoluted tubules) despite the presence of activated Notch1 in the nuclei of putative proximal progenitors. The inability of endogenous Notch1 to compensate for Notch2 deficiency may reflect sub-threshold Notch1 levels in the nucleus. In line with this view, forced expression of a gamma-secretase-independent form of Notch1 intracellular domain drives the specification of proximal fates where all endogenous, ligand-dependent Notch signaling is blocked by a gamma-secretase inhibitor. These results establish distinct (non-redundant), instructive roles for Notch receptors in nephron segmentation (Cheng, 2007).
Epithelial tubules consist of multiple cell types that are specialized for specific aspects of organ function. In the zebrafish pronephros, multiciliated cells (MCCs) are specialized for fluid propulsion, whereas transporting epithelial cells recover filtered-blood solutes. These cell types are distributed in a 'salt-and-pepper' fashion in the pronephros, suggesting that a lateral inhibition mechanism may play a role in their differentiation. The Notch ligand Jagged 2 is expressed in MCCs and notch3 is expressed in pronephric epithelial cells. Morpholino knockdown of either jagged 2 or notch3, or mutation in mind bomb (in which Notch signaling is impaired), dramatically expands ciliogenic gene expression, whereas ion transporter expression is lost, indicating that pronephric cells are transfated to MCCs. Conversely, ectopic expression of the Notch1a intracellular domain represses MCC differentiation. Gamma-secretase inhibition using DAPT demonstrated a requirement for Notch signaling early in pronephric development, before the pattern of MCC differentiation is apparent. Strikingly, it was found that jagged 2 knockdown generates extra cilia and is sufficient to rescue the kidney cilia mutant double bubble. These results indicate that Jagged 2/Notch signaling modulates the number of multiciliated versus transporting epithelial cells in the pronephros by way of a genetic pathway involving repression of rfx2, a key transcriptional regulator of the ciliogenesis program (Liu, 2007).
Previous studies have highlighted a role for the Notch signalling pathway during pronephrogenesis in the amphibian Xenopus laevis, and in nephron development in the mammalian metanephros, yet a mechanism for this function remains elusive. This study furthers the understanding of how Notch signalling patterns the early X. laevis pronephros anlagen, a function that might be conserved in mammalian nephron segmentation. The results indicate that early phase pronephric Notch signalling patterns the medio-lateral axis of the dorso-anterior pronephros anlagen, permitting the glomus and tubules to develop in isolation. This novel function acts through the Notch effector gene hrt1 by upregulating expression of wnt4. Wnt-4 then patterns the proximal pronephric anlagen to establish the specific compartments that span the medio-lateral axis. Pronephric expression was identified of lunatic fringe and radical fringe that is temporally and spatially appropriate for a role in regulating Notch signalling in the dorso-anterior region of the pronephros anlagen. On the basis of these results, a mechanism is proposed by which the Notch signalling pathway regulates a Wnt-4 function that patterns the proximal pronephric anlagen (Naylor, 2009).
Podocytes are highly specialized cells in the vertebrate kidney. They participate in the formation of the size-exclusion barrier of the glomerulus/glomus and recruit mesangial and endothelial cells to form a mature glomerulus. At least six transcription factors (wt1, foxc2, hey1, tcf21, lmx1b and mafb) are known to be involved in podocyte specification, but how they interact to drive the differentiation program is unknown. The Xenopus pronephros was used as a paradigm to address this question. All six podocyte transcription factors were systematically eliminated by antisense morpholino oligomers. Changes in the expression of the podocyte transcription factors and of four selected markers of terminal differentiation (nphs1, kirrel, ptpru and nphs2) were analyzed by in situ hybridization. The data were assembled into a transcriptional regulatory network for podocyte development. Although eliminating the six transcription factors individually interfered with aspects of podocyte development, no single gene regulated the entire differentiation program. Only the combined knockdown of wt1 and foxc2 resulted in a loss of all podocyte marker gene expression. Gain-of-function studies showed that wt1 and foxc2 were sufficient to increase podocyte gene expression within the glomus proper. However, the combination of wt1, foxc2 and Notch signaling was required for ectopic expression in ventral marginal zone explants. Together, this approach demonstrates how complex interactions are required for the correct spatiotemporal execution of the podocyte gene expression program (White, 2010).
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Biological Overview
| Regulation
| Protein Interactions | Post-transcriptional regulation of Notch mRNA
| Developmental Biology
| Effects of Mutation
| References
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