Vertebrate Development Laboratory, Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX, UK
* Author for correspondence (e-mail: julian.lewis{at}cancer.org.uk)
Accepted 9 November 2004
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SUMMARY |
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Key words: Notch, Inner ear, Chick, Hair cells, Electroporation, Lateral inhibition, Prosensory determination
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Introduction |
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Second, the components of the Notch pathway are expressed in a strongly
suggestive pattern (Fig. 1) in
the ear - the transmembrane receptor Notch ubiquitously (at least up to the
time of hair-cell specification), the transmembrane ligands Delta1 and
Serrate2 (Ser2, also known as Jagged2) in the nascent hair cells
(Adam et al., 1998;
Lanford et al., 1999
;
Morrison et al., 1999
;
Zine et al., 2000
), and
members of the Hairy/Enhancer of Split (Hes) family of transcription factors,
whose transcription is dependent on Notch activation, in the cells that are in
process of becoming supporting cells
(Lanford et al., 2000
;
Zheng et al., 2000
;
Zine et al., 2001
). This is
consistent with the idea that the nascent hair cells, by expressing Notch
ligands, activate Notch in their neighbours, thereby preventing the neighbours
from becoming hair cells too.
Third, defective Notch signalling in the ear of zebrafish
(Haddon et al., 1998;
Riley et al., 1999
) or mammals
(Kiernan et al., 2001
;
Lanford et al., 1999
;
Tsai et al., 2001
;
Zheng et al., 2000
;
Zine et al., 2001
;
Zine et al., 2000
) results in
excess production of hair cells. This is most strikingly exemplified in the
zebrafish mind bomb mutant (Jiang
et al., 1996
; Schier et al.,
1996
), in which Delta proteins fail to be ubiquitinated in the
normal way and are consequently unable to activate Notch
(Itoh et al., 2003
); as a
result, the cells of the inner ear sensory patches all differentiate as hair
cells, with none remaining as supporting cells
(Haddon et al., 1998
;
Haddon et al., 1999
). This
matches exactly the predictions of the classic lateral inhibition model.
There are, however, gaps in this evidence and reasons to think that the
full story cannot be so simple. The effects of loss of Delta1 in the mouse or
chick ear have not been reported, and homozygous Jagged2 mouse
mutants show only a mild excess of hair cells
(Lanford et al., 1999;
Zhang et al., 2000
).
Misexpression of Delta1 by means of a retroviral vector in the chick ear
failed to produce the expected disturbances of hair-cell production
(Eddison et al., 2000
).
Moreover, while the nascent hair cells express Delta1 and Ser2/Jagged2, the
precursor cells and supporting cells, more puzzlingly, also express a Notch
ligand, Ser1/Jagged1, and do so strongly and persistently
(Fig. 1). The loss of one copy
of Jagged1 in heterozygous Slalom
(Tsai et al., 2001
) and
Headturner (Kiernan et al.,
2001
) mutant mice, instead of causing overproduction of hair
cells, results in a mild reduction in their number in the cochlea, accompanied
by variable loss of the anterior and/or posterior cristae and semicircular
canals. The timing of gene expression in the normal embryo also hints that
Notch signalling does something more than the simple theory proposes. Notch
expression marks out the otic placode from surrounding ectoderm before it has
even begun to invaginate to form an otic vesicle, and expression of
Ser1/Jagged1 becomes visible soon after this, in a pattern that gradually
resolves into a set of discrete domains marking the sites of the future
vestibular and auditory sensory patches
(Adam et al., 1998
). This
suggests that Notch activity has some additional role at early stages.
In the present study, we have tested the effects of overactivating the Notch pathway in the embryonic chick inner ear, by using in-ovo electroporation of plasmid DNA to force transient expression of an activated form of Notch. We observed two consequences - one that we expected, and another that we did not: (1) within vestibular sensory patches, hair cell differentiation is inhibited; and (2) in parts of the inner ear that are normally non-sensory, ectopic sensory patches develop. These results indicate that Notch signalling has two quite different functions during inner ear development: initially, Notch activity in a group of cells makes them competent to form a sensory patch, conferring on them a prosensory character; subsequently, Notch-mediated lateral inhibition within such a patch governs the establishment of fine-grained patterns of differentiation of hair cells and supporting cells.
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Materials and methods |
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Electroporation of embryonic chick inner ear
Fertile White Leghorn eggs were incubated at 38°C. Embryos were staged
according to Hamburger-Hamilton (HH) tables. Microelectroporation of the inner
ear was performed at the otic cup stage (stages HH 13-17) as described in
Momose et al. (Momose et al.,
1999) with minor modifications, using a TSS-10 square-wave pulse
generator (Intracel, UK) to generate three 100-millisecond bursts of 30 Hz, 7
volt square-wave electric pulses. After electroporation, eggs were sealed with
tape and returned to incubation. The numbers of embryos analysed in detail for
each condition were as follows: NICD-IRES-GFP, 124;
HA-NICD-IRES-GFP, 98; IRES-GFP, 41. Following electroporation with
either construct, GFP protein was successfully detected by immunostaining in
the inner ear of approximately 70% of analysed specimens.
Embryos electroporated with NICD-IRES-GFP or HA-NICD-IRES-GFP were returned to the incubator for 1 (n=26), 2 (n=36), 3 (n=36), 4 (n=21), 5 (n=21), 6 (n=13), 7 (n=59), 8 (n=4) or 10 (n=6) days, then processed for further analysis. IRES-GFP controls were similarly analysed after 5 (n=11), 7 (n=25) or 10 (n=5) days.
Immunocytochemistry
Embryos were decapitated and their heads immersed in 4% paraformaldehyde in
PBS at 4°C for 2 to 12 hours. For whole-mount immunostaining, the
membranous part of the inner ear was dissected out from the surrounding
cartilage and incubated for 1 hour in PBS containing 0.3% Triton X100 and 10%
goat serum. All subsequent incubations and rinses were performed in PBS with
0.1% Triton X100 (PBT). Incubations with primary and secondary antibodies were
carried out in PBT for 2 hours at room temperature or overnight at 4°C.
Antibodies and reagents used were: rabbit serum anti-GFP (Molecular Probes,
1/2000), chicken IgY anti-GFP (Upstate, UK; 1/200; this antibody was used for
Ser1 and GFP double-immunostaining), rabbit serum anti-chicken Ser1 (1/50),
mouse anti-HA (Covance, UK; 1/500), mouse monoclonal IgG1 anti-HCA
(Bartolami et al., 1991)
(1/100), mouse monoclonal IgG2a anti-ß-III tubulin (TuJ1, Covance
Research, Cambridge Bioscience Ltd, UK; 1/1000), mouse monoclonal IgG2a
anti-HCS-1 (J. E. Finley and J. T. Corwin, unpublished)
(Gale et al., 2000
) (1/100),
Alexa A488-, A594-, and A633-conjugated secondary antibodies (Molecular
Probes, The Netherlands; 1/500 dilution), Cy2-conjugated donkey anti-chicken
IgY (Jackson ImmunoResearch, PA; 1/200), Alexa 633-conjugated phalloidin
(Molecular Probes; 1/100). Specimens were mounted in Slowfade (Molecular
Probes) and observed under a Zeiss LSM510 confocal microscope. For
cryosectioning, embryo heads were fixed as described above, then immersed in a
graded series of sucrose-PBS (5-10-20%), embedded in 1.7% agar with 5%
sucrose, frozen at -20°C, and sectioned at 15 µm thickness on a
Reichert-Jung cryomicrotome.
We used immunofluorescence, as opposed to intrinsic GFP fluorescence, for the detection of GFP in all the experiments described in detail in the Results.
Hair cell counts
The fraction of GFP-positive cells differentiating into hair cells was
estimated at E8.5-9.0 in vestibular sensory epithelium transfected at E2 with
either IRES-GFP (n=4 specimens) or NICD-IRES-GFP
(n=8 specimens). For each specimen, the number of GFP-positive hair
cells (expressing the Hair Cell Antigen) and the total numbers of GFP-positive
cells were counted within at least two 1000 µm2 regions in
vestibular sensory patches. The regions for counting were selected at random
subject to the requirement that each should contain at least 20 GFP-positive
cells and should be located in the central region of a vestibular sensory
patch. The values obtained for each condition were pooled, and used to
calculate the percentage of GFP-positive cells differentiating into hair
cells.
Whole-mount in-situ hybridisation
Whole-mount in-situ hybridisation was performed as described in
Ariza-McNaughton and Krumlauf
(Ariza-McNaughton and Krumlauf,
2002) with minor modifications, but the prehybridisation treatment
with proteinase K was omitted. Immunodetection of DIG-labelled RNA probes was
performed using an anti-DIG peroxidase antibody (diluted 1:100) and either the
TSA-FITC or the TSA-Cy3 amplification systems (Perkin Elmer).
For the timecourse analysis of GFP expression by in-situ hybridisation, embryos were electroporated with HA-NICD-IRES-GFP and then fixed at 1 (n=5), 2 (n=4), 3 (n=4), 4 (n=4), 5 (n=4) or 7 (n=8) days post-transfection.
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Results |
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An alternative approach is to incorporate the transgene into plasmid DNA
and introduce this into the cells in ovo by electroporation
(Muramatsu et al., 1997;
Nakamura and Funahashi, 2001
).
In this case, the time of onset of transgene expression is precisely
determined by the time of electroporation (beginning within 2 hours), and
subsequent transgene expression is limited in duration because the plasmid is
soon lost from the transfected cells.
We used this technique to transfect cells of the embryonic chick inner ear
at 2 to 3 days of incubation (stages HH 13-17) - the period in which the otic
placode invaginates to form first a cup and then a closed vesicle. At these
stages, the otic cup can be easily filled with a DNA solution and
electroporated efficiently without compromising embryo survival. To assess the
efficiency and timecourse of transgene expression, we used a control plasmid
containing an IRES-GFP sequence under a constitutive RSV promoter. More than
100 embryos were examined both alive and after sectioning at varying times
after electroporation. GFP fluorescence was directly visible in the otocyst
between 24 and 72 hours after electroporation, but was very much decreased or
absent in specimens examined 96 hours after transfection, suggesting that
transcription from the original plasmid had probably ceased by this time.
However, even small amounts of GFP protein could be detected by
immunochemistry, and we were able to find GFP-positive cells with this method
up to at least 10 days after electroporation. We could thus recognise
transfected cells and their progeny and assess their fate between E7 and 10,
when hair-cell differentiation is advanced and the hair cells are easily
distinguished by immunostaining for Hair cell antigen (HCA), a receptor-like
protein tyrosine phosphatase present in early differentiating hair bundles
(Bartolami et al., 1991;
Goodyear et al., 1995
;
Goodyear et al., 2003
), and/or
the HCS-1 antigen, an unidentified cytoplasmic protein specific to hair cells
(Gale et al., 2000
). At sites
of transfection within vestibular sensory patches and in the basilar papilla,
both hair cells and supporting cells were GFP-positive. The regular pattern of
differentiation of these two cell types was not altered by transfection with
the IRES-GFP vector (Fig.
2).
|
In 38 specimens (out of 59 analysed in eight separate experiments), groups of GFP-positive cells were detected within inner ear sensory patches. In 17 out of these 38 specimens, such GFP-positive cells were seen within vestibular patches and were then generally clustered together (contrasting with results of transfection with the control IRES-GFP construct, where the cells were more dispersed). These GFP-positive clusters displayed a greatly reduced density or complete absence of hair cells (Fig. 3A) compared with surrounding non-transfected tissue. For cases in which hair cells were seen within such GFP-positive regions (see region 2 in Fig. 3B), careful examination of z-series of optical sections revealed that these hair cells were either GFP-negative, or displayed much less GFP staining than their immediate neighbours (Fig. 3C). In fact, whereas in control specimens transfected with IRES-GFP alone 16.7% of GFP-positive cells (37 out of 222, range 7 to 35% for individual fields; n=8) differentiated into hair cells, only 1.2% of GFP-positive cells (19 out of 1554; range 0% to 5% for individual fields; n=19) did so in specimens transfected with NICD-IRES-GFP (see Materials and methods). These results provide direct confirmation that Notch activation within a sensory-patch cell inhibits it from differentiating as a hair cell.
|
Notch activity outside sensory patches can trigger ectopic hair cell formation
Unexpectedly, and in apparent contrast with the results just described, we
found that in a large proportion of the specimens electroporated with
NICD-IRES-GFP, groups of GFP-positive cells located outside the
limits of sensory patches contained HCA-positive cells (24 out of 38 specimens
analysed as whole-mounts showed such patches in vestibular regions, and 13 out
of 38 in the cochlear duct). These ectopic hair cells were sometimes
associated with abnormal outgrowths of the otic epithelium (see below); where
there was no such deformity, they were often located in the vicinity of
auditory and vestibular sensory patches, for example in regions such as the
epithelium flanking the inferior edge of the basilar papilla
(Fig. 4A,B) or at the external
border of cristae (Fig. 4C,D).
In some of the cases where small groups of cells exhibited high levels of GFP
immunoreactivity (Fig. 4D; Fig. 5C) it was possible to
confirm from the analysis of z-series of optical sections that the
ectopic hair cells displaying HCA-positive stereociliary bundles were
themselves GFP-positive. The ectopic sensory progenitor character of some of
the NICD-IRES-GFP transfected cells was confirmed by their
expression of the ß-III isoform of tubulin
(Fig. 5A) and of Bmp4
(Fig. 5B), two markers of
prosensory regions of the otocyst (Molea
et al., 1999; Wu and Oh,
1996
). The hair-cell specific marker HCS-1 antigen
(Gale et al., 2000
) was also
detected in some NICD-IRES-GFP transfected cells, supporting their
identification as ectopic hair cells, as shown in
Fig. 5C.
|
|
Although ectopic hair cells were detected in approximately 60% of transfected specimens, only a small fraction of GFP-positive regions within each specimen contained ectopic hair cells. In fact, we even found cases where two transfected patches were in very close proximity (Fig. 4E), and yet ectopic hair cells could be detected in only one of those patches. As we discuss below, this variability might reflect variation in the time for which plasmid-directed gene expression persisted in the transfected cells.
Serrate-1 is upregulated in NotchICD transfected cells
The results just described suggest that Notch activation in a group of
cells in the early otocyst can drive them to adopt a prosensory character, so
that they become competent to generate hair cells subsequently. From previous
work, it appears that expression of the Notch ligand Ser1 is a marker of this
state: the Ser1 gene is expressed in the chicken otocyst from as
early as E3.5 in a broad ventromedial domain spanning the future sensory
regions, and as ear morphogenesis proceeds, this expression domain resolves
into a number of discrete patches corresponding precisely to the regions in
which hair cells will develop (Adam et al.,
1998; Cole et al.,
2000
; Myat et al.,
1996
). We therefore looked to see whether Ser1 was expressed in
our ectopic NICD-induced sensory-like patches.
In specimens transfected with NICD-IRES-GFP at E2 and examined 5 or 7 days later, we found that Ser1 was indeed ectopically expressed in GFP-positive cells located outside the normal sensory patches (Fig. 6B). As noted previously for the occurrence of ectopic hair cells, however, ectopic Ser1 expression was only visible in a small fraction of such GFP-positive cells within each specimen. The levels of Ser1 immunoreactivity of transfected cells located within the normal sensory patches were not affected - they remained high (data not shown). In specimens examined at earlier stages, preceding hair-cell differentiation, expression of Ser1 in ectopic locations was detectable from 48 hours post-electroporation in transfected cells displaying high GFP levels (Fig. 6A), but was not seen at earlier time points (12 and 24 hours, results not shown). These results indicate that Notch activity in a patch of otic epithelium can induce Ser1 expression, one of the normal markers of future sensory-patch character.
|
To see how long transcription of NICD in fact persisted after electroporation, we used in-situ hybridisation to investigate the expression of GFP mRNA in the inner ear at various times after electroporation of the NICD-IRES-GFP plasmid (Fig. 7). Because in this construct, GFP and NICD coding sequences are transcribed as a single unit, the presence of GFP mRNA gives a direct indication of the presence of NICD mRNA. We found that GFP mRNA is present in large patches of cells in the otocyst at 24 hours after electroporation, but at 3 days after electroporation it is already restricted to smaller patches. At 7 days post-electroporation only a few scattered cells continue to express GFP mRNA; it is possible that these rare cells are those in which the plasmid DNA has been integrated into the host cell genome. At the same stage, however, GFP protein could be detected in a large number of cells in specimens processed for GFP immunostaining (compare Fig. 7 with Fig. 4); as expected, the protein persists and can be detected by a sensitive method after the mRNA has disappeared. These data support the notion that in the vast majority of transfected cells, transcription from the plasmid is transient and ceases within 3 to 7 days after electroporation as a consequence of cell division and plasmid degradation and/or dilution in the progeny of transfected cells. Electroporation experiments in the neural tube using the same plasmid but with an HA-tagged form of NICD led to similar conclusions, with NICD protein detectable in transfected cells at 1 and 2 days after electroporation at E2 but absent by 4 days (data not shown).
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Discussion |
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We suggest that the two effects represent distinct actions of Notch at different stages of development. At early stages, Notch activity drives cells into a prosensory state. Later, as cell differentiation begins, Notch signalling mediates lateral inhibition, whereby nascent hair cells within the prosensory patch inhibit their neighbours from differentiating as hair cells. We have argued that we are able to see and distinguish the two effects because, with our technique of transgenesis by electroporation, the exogenous NICD is expressed transiently, beginning early enough to exert the first, inductive, effect, and lasting long enough to exert the second, inhibitory, effect in some sensory patches but not in others.
Lateral inhibition mediated by Notch controls differentiation of hair cells and supporting cells
Previous studies of Notch signalling in the ear have focused almost
entirely on its role in controlling the choices cells make between hair-cell
and supporting-cell fates. As explained in the Introduction, there is
substantial evidence that Notch-mediated lateral inhibition is critical for
this process of cell diversification within sensory patches, but there are
also findings that conflict with the standard account of how the inhibition is
regulated.
To check the basic assumptions of the lateral inhibition model, therefore, we decided to use electroporation to misexpress NICD directly. According to the model, this should act within the transfected cells to prevent them from differentiating as hair cells. That is precisely the result we have obtained in the vestibular sensory patches. In the basilar papilla we did not see such an effect; but the basilar papilla differentiates 1 to 2 days later than the vestibular patches, and by this time gene expression from the electroporated plasmid has faded away. Thus we conclude that Notch activity within sensory patch cells is sufficient to prevent them from differentiating as hair cells - certainly in vestibular regions, and probably also in auditory regions.
In normal development, therefore, it seems that nascent hair cells must
somehow escape Notch activation. Even though they express Notch1 [initially at
least - see Adam et al. (Adam et al.,
1998)], and even though the Notch ligand Ser1 is expressed all
over the surfaces of the adjacent supporting cells
(Adam et al., 1998
), this
evidently fails to trigger production of NICD in the nascent hair
cells. How can this be? It is possible that members of the Fringe family of
glycosylases play a part here, as they are expressed in the sensory patches
(Cole et al., 2000
;
Morsli et al., 1998
;
Zhang et al., 2000
) and can
modify Notch to lessen its susceptibility to activation by Serrate
(Haines and Irvine, 2003
;
Hicks et al., 2000
). However,
the mouse Lunatic fringe knockout shows only very mild disturbances
of hair-cell production (Zhang et al.,
2000
). Another possibility is that nascent hair cells contain a
factor such as Numb that makes them immune to Notch activation
(Frise et al., 1996
;
Guo et al., 1996
;
Spana and Doe, 1996
). This
suggestion is consistent with observations of elevated Numb expression in hair
cells (Eddison et al., 2000
).
Alternatively, the supporting cells may lack a factor that is needed to make
Ser1 and any other Notch ligands they express functional, such as Mind bomb or
some related protein. Mechanisms postulating that cell fate choices are
governed by differences in the expression of co-factors of these types
(Schweisguth, 2004
) could
explain why simply overexpressing Delta1 has little effect on the pattern of
hair cell production (Eddison et al.,
2000
), even though signalling via Notch is critical.
An early phase of Notch activity promotes formation of prosensory patches
Our most novel finding is that Notch activity outside the normal sensory
patches can induce production of hair cells. These ectopic sensory cells were
unequivocally identified by the co-expression of two specific markers of hair
cells, the HCA and HCS-1 antigens. Further, some isolated ectopic hair cells
were clearly GFP-positive, suggesting that NICD-IRES-GFP acted
cell-autonomously to drive cells along the sensory pathway. These findings
seem at first to contradict those previously discussed, indicating that Notch
activity represses hair-cell differentiation. As we have explained, however,
the paradox is easily resolved if one postulates that Notch is acting in
different ways at early and late steps of sensory patch formation - early to
induce a prosensory state, and later to restrict hair-cell differentiation -
and that misexpression of its active form (NICD) is only transient
in our electroporation experiments. Although alternative interpretations are
possible - invoking, for example, spatial variations in NICD
activity - the explanation in terms of transient expression is well supported
by our timecourse data.
If Notch activity is indeed normally required for cells to adopt a
prosensory character, one would predict that an early blockade or reduction of
Notch signalling should hinder the specification of prosensory patches. Two
sets of observations give support to that prediction. Firstly, mice carrying
mutations of the Jagged1 gene frequently have, in addition to hair
cell patterning defects in the organ of Corti, missing or reduced anterior and
posterior semicircular-canal ampullae
(Kiernan et al., 2001;
Tsai et al., 2001
). Secondly,
the prediction tallies with the results of previous experiments in which we
used an RCAS viral construct to force expression of a truncated form of Delta1
(Delta1dn) that acts in Notch-expressing cells to make them
refractory to Notch activation (Henrique
et al., 1997
). Instead of provoking overproduction of hair cells,
as we originally expected, this resulted in many sites in which a patch of
RCAS infection containing no hair cells directly abutted a sensory patch
containing no RCAS-infected cells (Eddison
et al., 2000
). This is just what one would expect wherever the
RCAS infection happened to occur within a region normally destined to be
sensory: by blocking Notch activation and preventing adoption of a prosensory
character at this site, it would have created a boundary of the type that was
seen.
Notch receptors are expressed throughout the early otocyst, but presumably they are activated only in regions where Notch ligands are also present. From what we know of Ser1 expression, it seems that these regions in fact coincide with the future or actual sensory patches: as discussed above, Ser1 is expressed uniformly within prosensory patches well before the actual hair-cell and supporting-cell fate decisions occur. This implies that all sensory patch precursor cells experience some level of Notch activation during their developmental history. Our results argue that such early Notch activity has an essential role in the specification of sensory versus non-sensory regions of the otocyst.
The phenotype of the mind bomb zebrafish has an interesting
implication here. With its vast overproduction of hair cells at the expense of
supporting cells, this mutant shows a failure of lateral inhibition without
any failure of prosensory determination. The Mind bomb protein has been shown
to act on Delta proteins and to be required for their activity as Notch
ligands (Itoh et al., 2003);
but there is no evidence that Mind bomb is required for the activity of Ser
proteins. Combining the fish data with the chick data, it is therefore
tempting to suggest that activation of Notch by Ser1 (SerA in zebrafish) is
independent of Mind bomb, occurs early, and is specifically responsible for
prosensory determination, while activation of Notch by Delta1 is dependent on
Mind bomb, occurs later, and is specifically responsible for lateral
inhibition. Such distinct actions are not likely to reflect signalling
mediated by different Notch family members, as the chick appears to have only
two Notch genes (judging from a TBLASTN search of the current release of the
chick genome) and only one of these - Notch1 - is detectably expressed in the
embryonic ear (Myat et al.,
1996
) (data not shown).
There is a precedent for the prosensory effect of Notch signalling. Indeed,
studies in the Drosophila eye have revealed an exactly analogous
twofold function for Notch signalling, first in driving cells into a proneural
state and then, at a later stage, in mediating lateral inhibition to restrict
the proportion of these cells that differentiate as neurons
(Baker and Yu, 1997;
Li and Baker, 2001
). Moreover,
experiments in Drosophila (Kurata
et al., 2000
) and in Xenopus
(Onuma et al., 2002
) have
shown that ectopic expression of activated Notch can act at early stages to
induce expression of members of the Pax-Eya-Dach-Six gene network,
resulting in development of ectopic patches of eye tissue. Members of the same
gene network, or their homologues, are expressed in a localized fashion in the
otocyst and are implicated in the development of its sensory patches
(Abdelhak et al., 1997
;
Kalatzis et al., 1998
;
Laclef et al., 2003
;
Li et al., 2003
;
Ozaki et al., 2003
;
Xu et al., 1999
;
Zheng et al., 2003
). It will
be interesting to see whether the ectopic sensory patches we observe in our
experiments with NICD in the ear likewise reflect a role of
NICD as inducer of the expression of these genes.
Other signalling pathways are likely to cooperate with Notch to specify prosensory regions of the otocyst
Only a fraction of NICD-transfected cells in our experiments
gave rise to ectopic sensory patches containing hair cells. In part, this may
reflect the variation that is certainly present in the efficiency of
electroporation and in the initial number of plasmid copies per cell,
affecting both the level and duration of expression of NICD in
transfected cells. If NICD expression is too weak and brief, it may
not suffice for the initial induction of a prosensory patch; if it is too high
and prolonged, it may block the development of hair cells within it.
Regional differences in the competence of transfected cells to generate
ectopic sensory patches may also be important. Indeed, we noted that ectopic
patches tended to form more frequently in vestibular than in auditory regions,
and most often in the neighbourhood of the normal sensory patches. Many genes
encoding transcription factors or signalling molecules, including components
of the FGF, BMP, Hedgehog and Wnt pathways, are expressed in various
restricted domains of the early otocyst
(Barald and Kelley, 2004;
Fekete and Wu, 2002
;
Fritzsch and Beisel, 2003
;
Whitfield et al., 2002
). It is
thus likely that the cooperation of several signalling pathways is required
for the establishment of prosensory patches. The detailed regulatory
connections between these pathways and the expression and activation of Ser1
and Notch1 remain to be deciphered, but there seems to be a particularly close
relationship with the Wnt signalling pathway. Stevens et al.
(Stevens et al., 2003
)
activated this pathway ectopically in the chick ear, by infecting the early
otocyst with an RCAS retrovirus that carried a constitutively active form of
ß-catenin. This, like the ectopic expression of NICD, resulted
in development of ectopic sensory patches, although it also produced other
abnormalities, including gross malformations and changes of hair-cell
character, that we did not observe in our electroporation experiments. Thus
the Wnt and Notch signalling pathways in the ear may be linked in some sort of
regulatory cascade.
Notch activity stimulates expression of Ser1, creating a positive feedback loop
We have shown that ectopic NICD expression can induce ectopic
Ser1 expression, and this agrees with our previous finding that Ser1
expression is downregulated in cells where the Notch pathway is blocked by a
dominant-negative form of Su(H) (Eddison et
al., 2000). Positive regulation of Ser1 by Notch activity
also provides an explanation of some striking features of the normal Ser1
expression pattern. Unlike Delta1, Ser1 is expressed strongly and uniformly by
the cells in the prosensory patch, as though the cells are behaving
cooperatively instead of delivering lateral inhibition to one another. As
development proceeds, the boundary of the Ser1 expression domain becomes
sharply defined, and the expression of Ser1 within this domain persists,
continuing in the supporting cells even into adult life
(Stone and Rubel, 1999
), but
disappearing from the hair cells, in which Notch activity is absent. All these
features are to be expected, if Ser1 activates Notch and activated Notch
stimulates Ser1 expression. As we have discussed elsewhere
(Eddison et al., 2000
), this
positive feedback will give rise to lateral induction, as opposed to lateral
inhibition: a cell expressing Ser1 will tend to make its neighbours also
express Ser1, and the effect will be reciprocated. The positive feedback will
tend to intensify and perpetuate the expression of Ser1 and to sharpen the
boundaries between expressing and non-expressing regions. Against this
background of Ser1 regulation, other influences must act to allow a subset of
cells to escape Notch activation and differentiate as hair cells.
Conclusion
We have seen that Notch signalling in the ear involves two classes of
ligands, regulated in opposite ways and important for different effects. Ser1
is positively regulated by NICD, is expressed in each prosensory or
supporting cell, and, we suggest, acts on the neighbours of that cell to keep
them in a prosensory or supporting-cell state - that is, to maintain the
potential for future differentiation as a hair cell. Delta1 and Ser2 are
negatively regulated by NICD, are expressed in each nascent hair
cell, and act on its neighbours to prevent them from realizing their potential
to differentiate as hair cells. Both classes of ligands activate Notch1, the
only member of the Notch family that is expressed in the chick ear, as far as
we can tell. The difference of function between the ligands reflects the
difference in the way they are themselves regulated by NICD, and
the difference in the times at which they are called into play.
This picture is self-consistent and fits the data, but it leaves several mysteries unresolved. How are the nascent hair cells singled out? How do they, and they alone, avoid producing NICD, thereby freeing themselves of inhibition? Why is there such a delay from establishment of the prosensory patch to the onset of hair-cell differentiation? Why do only a fraction of NICD-IRES-GFP transfected cells differentiate into ectopic sensory patches? And what mechanism dictates in the first place where Notch is to be activated so as to drive cells into a prosensory state? Our demonstration of the two-fold action of Notch highlights these questions and takes us a step closer to answering them. What we discover for the ear will surely help to illuminate the workings of the Notch pathway in other developing and adult tissues, where similarly multiple Notch ligands are frequently deployed in time-dependent interlocking patterns.
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ACKNOWLEDGMENTS |
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