The Notch Ligand JAG1 Is Required
for Sensory Progenitor Development
in the Mammalian Inner Ear
Amy E. Kiernan, Jingxia Xu, Thomas Gridley*
The Jackson Laboratory, Bar Harbor, Maine, United States of America
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 p27kip1as 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 p27kip1are 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 p27kip1. These data demonstrate that JAG1-mediated Notch signaling is essential
during early development for establishing the prosensory regions of the inner ear.
Citation: Kiernan AE, Xu J, Gridley T (2006) The Notch ligand JAG1 is required for sensory progenitor development in the mammalian inner ear. PLoS Genet 2(1): e4.
The mammalian inner ear is a complex structure consisting
of a coiled cochlea, three orthogonally positioned semi-
circular canals, a central vestibule, and a dorsally projecting
endolymphatic duct and sac. With the exception of the
endolymphatic duct and sac, the different parts of the ear all
contain sensory organs populated by sensory hair cells and
their associated supporting cells. There are three different
categories of sensory organs: cristae, located at the base of
each semicircular canal; maculae, housed within the central
vestibule; and the organ of Corti, which lines the cochlear
duct. Only one sensory organ, the organ of Corti, is required
for hearing; the other five organs are important for balance.
Unfortunately, in mammals, if these regions are damaged due
to an environmental or genetic insult, they cannot regener-
ate, leaving a permanent hearing and/or balance impairment.
Although some progress has been made in understanding
how the individual cell types within the sensory areas of the
ear are formed [1,2], little is known about the molecular
mechanisms that establish the prosensory lineage and how
the different sensory organ types are formed. Interestingly,
the molecular mechanisms that underlie sensory differ-
entiation in the vertebrate inner ear demonstrate strong
parallels with Drosophila sense organ development [3–5]. For
example, during Drosophila external sense organ development,
lateral inhibition mediated by Notch signaling is required to
restrict the adoption of the sensory organ precursor cell fate,
which then gives rise to the entire sensory organ [6–8].
Similarly, in the vertebrate ear, lateral inhibition mediated by
Notch signaling appears to be important for restricting the
number of cells that can adopt the hair cell fate [9–15].
Lineage analysis has also shown that, at least in the chicken,
hair cells and supporting cells arise from a common
progenitor , consistent with an equipotent epithelium
that undergoes lateral signaling to specify cell fates. Unlike in
Drosophila, which has a single Notch receptor and two ligands
(Delta and Serrate/Jagged), in mammals Notch signaling
pathway components include four receptors (Notch 1–4)
and five ligands (Delta-like [DLL] 1, 3, and 4, and Jagged [JAG]
1 and 2; for reviews, see [8,17–19]). In the mouse, both DLL1
and JAG2 are expressed in nascent hair cells [10,20] and act
synergistically during lateral inhibition . Both DLL1 and
JAG2 appear to signal through the NOTCH1 receptor . In
Editor: David Beier, Harvard Medical School, United States of America
Received October 26, 2005; Accepted November 30, 2005; Published January 13,
A previous version of this article appeared as an Early Online Release on
December 1, 2005 (DOI: 10.1371/journal.pgen.0020004.eor).
Copyright: ? 2006 Kiernan et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Abbreviations: BMP, bone morphogenetic protein; cko, conditional knockout;
DLL, Delta-like; DSL, Delta-Serrate-Lag2; E[number], embryonic day [number]; ES,
embryonic stem; FGF, fibroblast growth factor; JAG, Jagged; SEM, scanning
* To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
PLoS Genetics | www.plosgenetics.orgJanuary 2006 | Volume 2 | Issue 1 | e4 0027
Drosophila, prosensory regions that undergo lateral inhibition
are first delineated by expression of members of the atonal or
acheate-scute family of basic helix-loop-helix transcription
factors. Further parallels with Drosophila have arisen with
the finding that an atonal homolog, the Math1 gene, is
required for hair cell differentiation [21–23]. However, the
situation is not entirely similar to Drosophila, as MATH1 does
not appear to be required to establish the prosensory regions
[22,24]. Instead, it has turned out that another type of
transcription factor, the HMG-box factor SOX2, is required
for sensory organ formation in the inner ear . This finding
does not demonstrate direct parallels with Drosophila sense
organ formation, since the Drosophila SOX2 homologs
SoxNeuro and Dicheate have no known role in peripheral sense
organ formation. Instead, both genes play a role in the
formation of neural progenitor cells in the central nervous
system [26,27]. Consistent with a prosensory role, SOX2
marks sensory progenitors early in development and acts
upstream of the Math1 gene during sensory organ formation
in the ear . Interestingly, one of the Notch ligands, JAG1,
is expressed early in the prosensory regions of the ear [4,20],
indicating that Notch signaling also may play a role in early
sensory organ formation. Consistent with this finding, mice
heterozygous for N-ethyl-N-nitrosourea-induced point muta-
tions in the Jag1 gene show mild sensory organ defects in the
ear [28,29]. Unfortunately, embryos homozygous for available
mutant alleles of the Jag1 gene do not survive past embryonic
day (E) 11.5 due to vascular defects, precluding an analysis of
their inner ears. To circumvent this early lethality, we have
created a conditional allele of the Jag1 gene using Cre/loxP
technology. Using the Foxg1-Cre mouse line to express Cre
recombinase in the early otocyst [30,31], we have disrupted
JAG1 function in the ear and show that sensory formation in
the inner ear is severely attenuated in these mutants. Analysis
of the patterns of hair and supporting cell formation in the
Jag1-conditional knockout (cko) inner ears suggests that fewer
progenitors form in Jag1-cko inner ears. This result is
confirmed by analysis of the prosensory markers SOX2 and
p27kip1, which are down-regulated as early as E12.5, indicat-
ing that the Jag1 gene acts early during sensory progenitor
formation. These data demonstrate an early role for Notch
signaling in establishing the sensory progenitors of the inner
Creation of a Conditional Allele of the Jag1 Gene
We created an allele for conditional inactivation of JAG1
function by flanking the Delta-Serrate-Lag2 (DSL) domain-
encoding exon (exon 4) of the Jag1 gene with loxP sites
(Figure 1). The DSL domain has been shown to be the region
of the DLL and JAG proteins that interacts with Notch family
receptors [32,33]; therefore, removing this region of the gene
should create a nonfunctional protein. To demonstrate that
this allele encodes a nonfunctional protein, we crossed
Jag1flox/þ mice to mice expressing Cre recombinase in the
female germline under control of the Zp3 promoter (Zp3-Cre
mice); the Zp3-Cre mouse strain has been shown to express
Cre recombinase in the growing oocyte prior to the
completion of the first meiotic division . Female Zp3-Cre/
þ; Jag1flox/þ offspring were then crossed to male B6 mice to
produce offspring that were heterozygous for the deleted
region of the floxed allele (designated Jag1del2/þ; see Materials
and Methods). Heterozygous Jag1del2/þmice were intercrossed,
and Jag1del2/Jag1del2homozygous offspring were analyzed for
defects between E9.5 and E11.5. Jag1del2/Jag1del2mutant
embryos exhibited the same vascular phenotype we described
previously in embryos homozygous for a targeted Jag1 null
allele, Jag1del1. Specifically, the Jag1del2/Jag1del2mutant
embryos exhibited yolk sac vascular remodeling defects and
cranial hemorrhaging, and often exhibited an enlarged
pericardial sac (Figure 2A–2D). All Jag1del2/Jag1del2mutant
embryos were necrotic by E11.5, and most showed vascular
defects by E10.5. RT-PCR of cDNA synthesized from the
mutant embryos using primers that span the floxed exon 4
demonstrated that this region was deleted, as expected
(Figure 2E). These data demonstrate that deletion of the
Jag1floxallele yields a nonfunctional Jag1 mutant allele.
Inactivation of Jag1 Function within the Ear
To disrupt JAG1 function within the inner ear, we crossed
Jag1flox/Jag1floxmice with mice doubly heterozygous for the
Foxg1-Cre allele (these mice express Cre recombinase through-
out the otocyst, as well as forebrain, eye, and foregut)  and
the Jag1del1allele . Offspring with the genotype Foxg1-Cre/þ;
Jag1del1/Jag1flox(hereafter designated Jag1-cko) survived through
E18.5 and were analyzed for inner ear defects. We examined
the patterns of Cre-mediated excision in Jag1-cko embryos at
E10.5 and in cochleae at E16.5–E18.5 by in situ hybridization
using a probe that specifically detected the deleted exon 4
(Figure 3). These results showed that expression in the otocyst
was weak or absent by E10.5 (Figure 3B). In addition, analysis
of cochlear expression at later stages showed no expression at
E16.5 (Figure 3F) and E18.5 (unpublished data). These data
indicate that, as previously shown for conditional deletion of
a Fibroblast growth factor receptor 1 (Fgfr1) floxed allele , the
Foxg1-Cre line efficiently deletes the Jag1floxallele early during
inner ear development.
Malformation of the Inner Ear in Jag1-cko Mutants
To examine the morphology of the Jag1-cko inner ears,
paintfilling of the inner ears of mutants and controls was
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JAG1 Function in Ear Sensory Development
Deafness and adult-onset hearing loss are significant health
problems. In most cases, deafness or vestibular dysfunction results
when the sensory cells in the inner ear, known as hair cells,
degenerate due to environmental or genetic causes. In the
mammalian inner ear, the hair cells and their associated supporting
cells can be found in six different patches that have particular
functions related to hearing or balance. Unfortunately, unlike in
birds or fish, mammalian hair cells show little ability to regenerate,
resulting in a permanent hearing or balance disorder when
damaged. Here, the authors show that a protein called JAG1, a
ligand in the Notch signaling pathway, is required for the normal
development of all six sensory regions in the mammalian inner ear.
In ears that lacked JAG1, some of the sensory patches were missing
completely, whereas others were small and lacked particular cell
types. The authors showed that JAG1 is required by the sensory
precursors, progenitor cells that give rise to both the hair cells and
the supporting cells. By understanding how the sensory areas
develop normally, it is hoped that molecular tools can be developed
that will aid sensory regeneration in the mammalian inner ear.
performed at E15.5 (Figure 4). Results of this analysis showed
a severe disruption in the structure of the Jag1-cko inner ears
compared to their littermate controls (Figure 4C and 4D).
Specifically, the semicircular canals were largely absent, with
the exception of a portion of the anterior and lateral
semicircular canals. In addition, the utricle appeared small,
the saccule was misshapen, and the cochlea was undercoiled.
In contrast, the parts of the inner ear that are not associated
with sensory formation, including the endolymphatic duct
and sac and the common crus, appeared relatively unaffected.
Sensory Defects in Jag1-cko Mutant Inner Ears
Since the Jag1 gene is expressed in the sensory areas of the
ear, and because the structural malformations observed in
Jag1-cko mutant inner ears appeared to primarily affect
regions of the ear that contained sensory organs, we
examined the sensory regions of the ear for defects. We
examined the organ of Corti, the sensory organ of the
cochlea, at E18.5 by scanning electron microscopy (SEM)
(Figure 5). By this stage, all hair cells within the organ of Corti
have exited the cell cycle, and most are well-differentiated
although not fully mature . Severe hair cell patterning
defects were apparent by SEM within the Jag1-cko mutant
cochleae. This phenotype was most striking in the basal turns
of the cochlea, where no hair cell formation was observed
(Figure 5D). In the midbasal regions of the organ of Corti,
hair cells formed in patches, within which there was no clear
formation of rows or distinction between inner and outer
hair cells (Figure 5F). More apically, hair cells appeared more
continuous along the organ of Corti (Figure 5H). However,
although hair cells were present in the apical region, their
Figure 1. Construction of a Conditional Allele of the Jag1 Gene
(A) Schematic diagram showing the strategy for generating Jag1floxmice. The targeting vector was designed to insert loxP sites (black arrowheads) on
either side of exon 4, the DSL domain-encoding exon (white area in exon 4). The neomycin resistance cassette (for positive selection) was flanked by FRT
sites (gray arrowheads) so that it could later be removed by crossing to FLPe-expressing mice. A diphtheria toxin gene was included for negative
selection. Dotted lines depict the recombination events that occur when either the FLPe or Cre recombinases are present. Primer positions for
genotyping are shown as small black arrows (a, DSLF; b, J1LoxR1; c, J1FlpF1; see Materials and Methods for sequences). DT, diphtheria toxin; R, EcoR1; X,
(B) Southern blot analysis of EcoRI-digested DNA from ES cells using the external probe shown in (A). Left lane shows the wild-type band (12.3 kb), and
the center and right lanes show correctly targeted ES cells that have both a wild-type band and a smaller mutant band (9.4 kb).
PLoS Genetics | www.plosgenetics.orgJanuary 2006 | Volume 2 | Issue 1 | e40029
JAG1 Function in Ear Sensory Development
numbers were clearly reduced; rather than the normal,
perfectly ordered four rows of hair cells, there were only
two rows of loosely arranged hair cells of indistinct type.
Abnormal Hair and Supporting Cell Patterns in Jag1-cko
To determine which sensory cell types were differentiating
in the Jag1-cko mutant cochleae, specific markers were used to
identify hair cell and supporting cell subtypes throughout the
ear (Figure 6). In the cochlea, we used an antibody against
MYO7A to label all hair cells and an antibody against S100A1
to label inner hair cells, Deiter’s supporting cells, and inner
phalangeal supporting cells . When both markers were
used in combination, inner hair cells, outer hair cells, and
some supporting cell types could be distinguished (Figure
6A–6F). This analysis showed that in the apex of the cochlea,
inner hair cells were present and usually formed as doublets
(Figure 6B). Their associated supporting cells, the inner
phalangeal cells, were also present. Outer hair cells and their
associated supporting cells, the Deiter’s cells, were not
present in this region. In the middle portions of the cochlea,
both inner and occasionally outer hair cells were present,
although their patterning was clearly abnormal (Figure 6D).
In addition, the tunnel of Corti was not apparent, and there
were often doublets of inner hair cells and increased numbers
of outer hair cell rows without accompanying Deiter’s
supporting cells. As shown by SEM, both hair cells and
supporting cells were absent in the very basal regions of the
cochlea (Figure 6F).
Using the same markers we also examined the vestibular
sensory organs in Jag1-cko mutant inner ears (Figure 6G–6J).
Consistent with the lack of semicircular canal and ampulla
Figure 2. Jag1del2/Jag1del2Embryos Exhibit Vascular Defects and Lethality
Consistent with Loss of JAG1 Function
(A and B) E10.5 embryos demonstrating the loss of large blood vessels
(white arrows in [A]) in the Jag1del2/Jag1del2yolk sacs (B) similar to other
Jag1 loss-of-function mutants. (C and D) E11 embryos demonstrating a
small, necrotic Jag1del2/Jag1del2embryo (D). White arrow in (D) indicates
an enlarged pericardial sac (enlarged, inset), which is frequently
observed in mutants exhibiting cardiovascular defects. RT-PCR results
using primers that span exon 4 using RNA extracted from E10.5 control
(þ) and ZP3-Cre deleted embryos (?) (E). The upper band (541 bp)
indicates that the wild-type allele is present. The lower bands (286 bp)
indicates the Jag1del2mutant allele that does not contain exon 4.
Figure 3. Conditional Jag1 Inactivation Using the Foxg1-Cre Line
(A–D) Low- and high-power views of E10 embryos processed for whole-
mount in situ hybridization using a Jag1 exon 4-specific probe. White
arrows (A) point to the Jag1 signal in the otocyst (left arrow) and the eye
(right arrow), two structures where Cre recombinase is expressed. In
Jag1-cko mutants at E10, this signal is either absent or extremely weak.
Black arrows (A and B) point to expression in the spinal cord and nephric
duct, regions where Cre recombinase expression has not been reported
in Foxg1-Cre mice. However, expression is consistently weaker in these
areas in Jag1-cko embryos, indicating that there may be low levels of
widespread expression of Cre recombinase in Jag1-cko embryos. In (D),
the otocyst and the eye are outlined by a dotted line. Very little
expression is observed in these regions, consistent with Foxg1-Cre
(E and F) In situ hybridization of E16.5 cochleae demonstrating Jag1
expression in wild-type (E) and Jag1-cko cochleae (F), where expression is
entirely absent. Scale bars ¼ 500 lm.
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JAG1 Function in Ear Sensory Development
formation observed by paintfilling, there was no evidence of
crista formation. The Jag1-cko utricular macula was extremely
small with very few differentiating hair cells (Figure 6H).
Surprisingly, the saccule and its macula were only mildly
affected in the Jag1-cko inner ears (Figure 6J). Hair cell
differentiation appeared relatively unaffected, although the
entire saccular structure was shaped differently than in the
controls, a feature that was also observed in the paintfilled
specimens (see Figure 4C and 4D). These data show that all
sensory organs within the inner ear are affected to varying
degrees in Jag1-cko inner ears. However, some sensory organs,
such as the cristae, appear to be more sensitive to the loss of
To examine whether aberrant hair cell patterning in Jag1-
cko cochleae was due to defects in hair cell formation or in
subsequent differentiation, we examined hair cell patterning
at an earlier stage (E16.5). Using a lectin that binds to hair cell
stereocilia, we examined whether the patterns of hair cell
formation at E16.5 looked similar to the patterns at E18.5
(Figure 7). At E16.5 in wild-type cochleae, a gradient of hair
cell differentiation was evident (Figure 7A, 7C, 7E, and 7G); in
the basal regions both inner and outer hair cells could be
recognized (unpublished data), while in the middle regions
only inner hair cells were clearly detected by most markers
(Figure 7A and 7C). In the more apical regions, little to no
hair cell differentiation had taken place by this stage (Figure
7E and 7G). In Jag1-cko cochleae, the patterns looked similar
to those at E18.5, with patches of hair cells in the midbasal
regions (Figure 7B and 7D) and a complete absence of hair
cells in the very basal regions (Figure 7B). These data suggest
that the Jag1-cko mutants have defects in hair cell formation
rather than differentiation. In addition, the apical regions in
the Jag1-cko cochleae did not appear more differentiated than
the controls (Figure 7E–7H), arguing against precocious
differentiation as an explanation for the reduced numbers
of hair cells observed in the mutant cochleae.
Disrupted Prosensory Development in Jag1-cko Inner Ears
To determine how the JAG1 ligand functions during
sensory development, we used several markers of the
Figure 5. Hair Cell Patterning Defects in the Cochlea
Scanning electron micrographs demonstrating the different patterns of
hair cell production along the length of the cochlea in Jag1-cko embryos.
(A–D) Low-power views of the apical and basal cochlear turns. The
boxed-in area along the base in (A) and (B) is shown at higher
magnification in (C) and (D). Note the absence of hair cells in the base of
the Jag1-cko cochlea, except for a small patch of cells in the more apical
portion (arrow). Scale bars ¼ 500 lm.
(E and F) In the midbasal region, more hair cells are observed, but they
are arranged in patches, with no clear distinction between inner and
outer hair cells.
(G and H) In the apical turn, hair cells are continuous but generally
arranged in only two rows. Scale bar ¼ 100 lm.
Figure 4. Inner Ear Dysmorphology in Jag1þ/?and Jag1-cko Embryos
E15.5 inner ears that have been paintfilled to display their overall
(A) Wild-type inner ear showing normal morphology. Structures are
labeled as follows: aa, anterior ampulla; asc, anterior semicircular canal;
cd, cochlea duct; ed, endolymphatic duct; la, lateral ampulla; lsc, lateral
semicircular canal; pa, posterior ampulla; psc, posterior semicircular
canal; sac, saccule; ut, utricle.
(B) Jag1 heterozygote inner ears (either Jag1del1/þ or Foxg1-Cre/þ;
Jag1flox/þ) display truncated posterior semicircular canals and missing
ampullae (asterisk). Arrows point to the anterior and posterior ampullae,
which are small compared to the wild-type control (A).
(C and D) A much more severe phenotype is observed in Jag1-cko
animals. There are no ampullae and little semicircular canal develop-
ment; an asterisk indicates a remnant of the anterior canal. In (D), there is
also a remnant of the lateral canal (arrow). The utricle and saccule are
smaller and the cochleae are shorter and undercoiled. Scale bar¼500lm.
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JAG1 Function in Ear Sensory Development
prosensory domain, including p27kip1and SOX2, and
examined their expression patterns in both wild-type and
Jag1-cko mutant cochleae (Figure 8). At E14.5, the majority of
hair cells and supporting cells in the organ of Corti have
completed their final division, and hair cells are beginning to
differentiate in the basal portions of the cochlea . p27kip1,
a cell-cycle inhibitor, is required for the cochlear sensory
progenitors to exit the cell cycle on time, and is an
established marker of the prosensory domain in the cochlea
[22,37]. p27kip1begins to be expressed in a discrete domain
within the cochlea as the hair cells and supporting cells exit
the cell cycle around E13.5 to E14.5 (Figure 8A, 8B, 8D, and
8E). Recently it has been shown that the SRY-related
transcription factor SOX2 is required for establishment of
the prosensory regions in the inner ear . Using fluo-
rescence immunocytochemistry double labeling, we exam-
ined the relationship between these markers and JAG1
protein expression in both wild-type and Jag1-cko cochleae.
As previously reported , JAG1 was not expressed within
the prosensory domain as assessed by p27kip1expression at
E14.5, but instead was expressed immediately adjacent
(possibly with some slight overlap) in the inner (neural)
portion of cochlea (Ko ¨lliker’s organ; Figure 8A and 8D). In
contrast, SOX2 did show a largely overlapping domain with
p27kip1(Figure 8B and 8E), as originally described .
However, the SOX2 expression domain was slightly larger
than the p27kip1domain, extending into Ko ¨lliker’s organ and
overlapping with the JAG1 domain. Despite the fact that
JAG1 was not expressed within the prosensory domain at
E14.5, both p27kip1and SOX2 expression was absent in the
basal regions of the cochlea (Figure 8C), indicating that
prosensory formation is already disrupted in these ears. In
the apex, weak expression of both markers was observed
(Figure 8F), consistent with the fact that some sensory
differentiation occurs in this region of the Jag1-cko cochlea.
In order to determine if JAG1 is ever expressed in the
prosensory region of the cochlea, we examined an earlier age
(E12.5) and compared the JAG1 domain to the SOX2 domain
(since p27Kip1is not expressed in the inner ear prior to E13.5
to E14.5). Adjacent sections from both wild-type and Jag1-cko
cochleae were immunostained to detect either JAG1 or SOX2
protein (Figure 9). This analysis showed that in the basal
regions of the wild-type cochlea, where sensory progenitors
were still dividing, JAG1 expression did overlap with the
SOX2 domain (Figure 9A and 9B), indicating that JAG1 is
initially expressed within the prosensory domain. However,
in the apical regions, where the sensory precursors have
ceased dividing, expression of JAG1 and SOX2 did not
overlap (Figure 9D and 9E). In the Jag1-cko cochlea, SOX2 was
absent from the basal regions and significantly down-
regulated in the apical regions (Figure 9C and 9F). These
data demonstrate that JAG1 is expressed within the prosen-
sory domain of the cochlea at early stages, and that, in the
absence of JAG1 function, sensory formation is disrupted
prior to cell cycle exit and differentiation of sensory hair cells
and nonsensory supporting cells.
We also compared JAG1 and SOX2 expression in the
vestibular regions of the inner ear in both wild-type and Jag1-
cko mutant embryos. JAG1 and SOX2 exhibited largely
overlapping expression domains that corresponded to the
locations of the five sensory organs in the vestibular portion
of the ear (Figure 10). The two expression domains only
Figure 6. Hair and Supporting Cell Markers Demonstrate Sensory Areas Are Reduced or Absent in the Jag1-cko Inner Ear
Immunocytochemistry using two markers, myosin VIIA (red; all hair cells) and S100a (green; inner hair cells, Dieter’s cells, and inner phalangeal cells)
demonstrate patterns of hair and supporting cell production at E18.5 in control and Jag1-cko inner ears.
(A–F) Sections through the indicated turns of the cochlea. Note the different hair cell patterns in the apical, middle and basal turns of the Jag1-cko
cochlea. Normal morphology is shown (A) along with labeled structures, as follows: GER, greater epithelial ridge; IHCs, inner hair cells (color-coded
yellow); LER, lesser epithelial ridge; OHCs, outer hair cells (color-coded red); SCs, supporting cells (color-coded green).
(G–J) Patterns of hair and supporting cell production in the vestibular system. The utricular macula is extremely small with very few hair cells (H) while
the saccule (J) shows robust hair and supporting cell production although the shape of the organ is smaller and malformed.
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JAG1 Function in Ear Sensory Development
differed significantly in the anterior and posterior cristae,
where JAG1 expression had a negative patch in the middle of
its expression domain, whereas SOX2 expression did not
show this same patch (Figure 10A, 10B, 10G, and 10H). The
JAG1-negative region may correspond to the eminentia
cruciatum, a nonsensory region present in the middle of
both the anterior and posterior cristae, although it is not
clear why SOX2 would be expressed there. In the Jag1-cko
vestibular sensory patches, SOX2 expression was consistent
with the patterns of sensory differentiation observed at E18.5.
For example, the Jag1-cko saccule displayed fairly normal
SOX2 expression (Figure 10C), consistent with the almost
normal development of the saccular macula. In contrast,
SOX2 expression in the utricle was very weak and the
expression domain was much smaller than in controls (Figure
10F), consistent with the severe disruption of differentiation
of the utricular macula in Jag1-cko inner ears. There was no
SOX2 expression in the Jag1-cko cristae, and in fact the entire
ampullae appeared to be missing or severely disrupted even
at this early stage (Figure 10C and 10I; dotted line regions),
consistent with the lack of cristae and ampullae observed at
We have demonstrated that Notch signaling, mediated by
the JAG1 ligand, is required early in development for the
formation of the sensory regions of the ear. By comparing
expression of JAG1 to two markers of the prosensory domain,
SOX2 and p27kip1, we have shown that JAG1 marks all
prosensory regions of the ear from early time points (E12.5),
but becomes down-regulated in the organ of Corti by E14.5,
when the sensory progenitors exit the cell cycle and begin
differentiating into hair cells and supporting cells. Both
SOX2 and p27kip1are down-regulated in the affected
prosensory regions of the Jag1-cko inner ear, demonstrating
that JAG1 is necessary for the development of early sensory
progenitors in the inner ear.
Distinctive Patterns of Hair Cell Formation in Jag1-cko
Inner Ears Suggest Progenitor Cell Numbers Are Reduced
One intriguing result from our studies was that the six
sensory regions were not equally affected by the loss of Jag1
function. For example, in the Jag1-cko vestibular system, the
cristae were lacking altogether, and only a small number of
hair cells differentiated in the utricular maculae. In contrast,
the saccular maculae exhibited little disturbance in hair cell
formation, although the overall shape of the organ was
abnormal. In the Jag1-cko cochlea, hair cell differentiation
patterns varied based on their apical or basal location. For
example, in the apical regions of the cochlea only inner hair
cells formed, and these were often arranged in multiple rows
rather than the normal single row. In the middle and
midbasal turns of the cochlea, patches of hair cells with
nonsensory intervening regions were frequently observed.
Within these patches, outer hair cells were sometimes
present, although the patterning was abnormal and S100A1-
labeled Dieter’s cells were not present. In the very basal
regions of the cochlea, neither hair cells nor supporting cells
The patches of hair cells found in the basal regions of the
cochlea and the differential effect of the mutation on the
basal and apical portions of the cochlea were particularly
interesting, as similar defects have been found in at least two
other mouse mutants of genes known to play a role in the
generation of the sensory precursors of the ear. For example,
both a hypomorphic allele and a conditionally deleted allele
of the Fgfr1 gene exhibited patches of hair cells in portions of
the cochlea . Similar to the Jag1-cko phenotype, these
patches in the Fgfr1 conditional mutants contained mostly
inner hair cells that were often arranged in multiple rows,
with very few outer hair cells. Unlike the Jag1-cko phenotype,
Fgfr1 function was required only in the cochlea. Another
mouse mutant, a hypomorphic allele of the Sox2 gene (yellow
submarine; Sox2ysb), also displayed patches of hair cells in the
basal portions of the cochlea and a milder phenotype in the
apical regions of the cochlea . More similar to the Jag1-cko
Figure 7. Early Analysis of the Patterns of Differentiation in the Jag1-cko
Cochlea Indicates the Defects Are Caused by a Failure in the Formation
of Sensory Cells and Not Subsequent Degeneration
Lectin staining of whole-mount cochlea at E16.5.
(A) Normal patterning in wild-type control cochlea. GER, greater
(B) Both the basal and middle portions of the cochlea are shown,
although because it is much longer in the control (A), the very basal
portion of the cochlea has been removed. Note the lack of hair cells in
the basal portion of the Jag1-cko cochlea.
(C–H) Boxed-in areas of (A) and (B) are shown at higher magnification in
(C) and (D). Arrows in (D) indicate the abnormal patches of hair cells also
observed at E18.5. Similarly, the boxed-in regions of (E) and (F) are shown
at higher magnification in (G) and (H), demonstrating the few hair cells
that are just beginning to differentiate in this region in both the control
and the mutant (arrowheads). Scale bars¼500 lm for the corresponding
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JAG1 Function in Ear Sensory Development
phenotype, SOX2 was required for both the cochlear and
vestibular sensory regions . The finding that primarily
inner hair cells differentiate in these mutant cochleae may be
due to the fact that inner hair cells are the first to
differentiate [10,37], suggesting that if there are reduced
numbers of progenitor cells, they would likely differentiate as
inner rather than outer hair cells. Similarly, the milder
phenotype in the apical regions of these mutants may be due
to the fact that cells exit the cell cycle earliest in the apex .
This may mean that, if there are reduced numbers of
progenitor cells, they would reside in the apical rather than
the basal portions of the cochlea.
The multiple rows of inner hair cells observed in Jag1-cko
and other mutants could be explained by a number of
different scenarios. One possibility is that the multiple rows
are not a result of actual increases in inner hair cell numbers,
but rather are caused by defects in their eventual arrange-
ment due to the shorter Jag1-cko cochlea. Recent studies of
mouse mutants with defects in planar cell polarity and
convergent extension (a term referring to the intercalation of
cells, leading to growth of tissue in one dimension in the
absence of proliferation) indicate that multiple rows of hair
cells can be obtained in this way and are frequently observed
in the apical regions of the cochlea [38–41]. An alternative
possibility is that the multiple rows are a result of a second,
later function of the JAG1 ligand, distinct from its prosensory
role described here. A third possibility is that the down-
regulation of p27kip1, a protein that inhibits continued
proliferation of the precursor cells in the cochlea, leads to
continued cell division of the remaining sensory progenitors,
ultimately resulting in excess numbers of inner hair cells in
the regions where they form. Taken together, these data
suggest that sensory progenitors are reduced in the Jag1-cko
inner ears and that Notch signaling, fibroblast growth factor
signaling, and the transcription factor SOX2 all act in either
common or parallel pathways involved in the production of
sensory progenitors in the inner ear.
A Prosensory Role for Notch in the Ear
An examination of early prosensory markers, including
p27Kip1and SOX2, demonstrated that prosensory establish-
ment is disrupted in Jag1-cko inner ears, consistent with the
suggestion that progenitors are reduced in these mutants.
Our data show that JAG1 plays an early prosensory role in ear
development, quite unlike the role played later during
development by the other Notch ligands, DLL1 and JAG2,
which are involved in lateral signaling and differentiation
[10,15]. These data are consistent with an early role for Notch
signaling in progenitor cell maintenance in the inner ear. In a
number of other systems, including the nervous system and
more recently in the intestinal epithelium, it has been
demonstrated that Notch signaling is involved in maintaining
cells in an undifferentiated state [42–46]. In the mammalian
nervous system it has been shown that loss of Notch signaling
leads to premature differentiation and a reduction in the
progenitor pool . Consistent with these findings, in vitro
studies have demonstrated that the frequency of neurosphere
production was reduced in Notch signaling mutants [47,48],
indicating a loss of stem cell potential. Moreover, studies have
also shown that Notch signaling promotes radial glial identity,
a cell type that has been shown to act as a progenitor cell in
the central nervous system [49–52]. Our results suggest that,
similar to the nervous system, Notch signaling via JAG1 is
important for sensory precursor formation or maintenance
in the inner ear. However, unlike the nervous system, we see
no evidence for precocious differentiation, suggesting instead
that JAG1 may affect the specification, survival, or prolifer-
ative capacity of the sensory precursors.
Recent evidence from the chick indicates that JAG1 may be
important for the initial sensory specification events. By
expressing a constitutively active form of Notch (Notch1-
ICD), Daudet et al.  demonstrated that ectopic sensory
patches could be induced, indicating that early Notch
signaling may be important for the induction of sensory
areas, and not just for their maintenance. However, it should
Figure 8. At E14.5 JAG1 Is Expressed Directly adjacent to the Prosensory Domain That Is Disrupted in Jag1-cko Inner Ears
Immunocytochemistry at E14.5 using two markers of the prosensory domain, Sox2 and p27Kip1, in combination with JAG1 in both the basal and apical
turns of the cochlea. Note that the JAG1 domain (red) does not overlap the p27Kip1domain (green) (A and D), whereas the SOX2 domain does largely
overlap with p27Kip1(yellow) (B and E). Both SOX2 and p27Kip1are down-regulated in the Jag1-cko cochlea (C and F), although there is weak expression
of both markers in the apex (F). GER, greater epithelial ridge; LER, lesser epithelial ridge.
PLoS Genetics | www.plosgenetics.orgJanuary 2006 | Volume 2 | Issue 1 | e40034
JAG1 Function in Ear Sensory Development
be noted that ectopic sensory areas formed only in certain
areas of the ear, indicating that some sensory competence is
required for this effect. A similar result was obtained by
overexpressing an activated form of b-catenin, an essential
component of the canonical Wnt signaling pathway, in the
chicken inner ear . As in the Notch1-ICD studies, ectopic
sensory regions were obtained, but again, only in certain
regions of the ear. However, unlike the Notch gain-of-
function studies, overexpression of b-catenin also led to a
change in sensory region character (i.e., cochlear to vestib-
ular), indicating that Wnt signaling governs not only whether
a sensory region will form but also the type of sensory region
that will form. In Drosophila, interactions between Notch and
Wingless, a member of the Wnt family of signaling molecules,
are well established [54,55], and evidence of an interaction
has begun accumulating in vertebrates as well [45,56,57]. Bone
morphogenetic protein (BMP) signaling may also be impor-
tant for sensory formation, particularly for the sensory
cristae, as BMP4 has been shown to mark the mouse cristae
from very early in development . Experiments in the
chicken have shown that blocking BMP signaling sometimes
leads to disturbances in sensory development . Taken
together, these data indicate that, based on expression
patterns, previous studies, and the evidence presented here,
JAG1 is the ligand responsible for the prosensory function of
the Notch pathway in the ear. Furthermore, the Notch
pathway likely interacts with other signaling pathways such as
the Wnt, FGF, and BMP pathways to create sensory organs of
the proper size, organization and character.
Sensory Formation Still Occurs in Jag1-cko Inner Ears
One somewhat puzzling question is that, if JAG1 is
important for sensory progenitor development, why does
any sensory formation occur in Jag1-cko inner ears? One
possibility is that another Notch ligand is compensating for
the loss of JAG1 function. This explanation seems unlikely
since none of the other Notch ligands shows a similar
expression pattern to JAG1 in the ear. For example, both the
Dll1 and Jag2 genes are expressed in nascent hair cells after
they exit the cell cycle and begin differentiating. However, in
addition to hair cell expression, there is also early expression
of the Dll1 gene in the anteroventral portion of the otocyst at
about E10.5 [4,20], that likely overlaps with at least part of the
JAG1 domain (see Figure 2) . This expression domain has
previously been thought to be related to the formation of the
neuroblasts that delaminate from the otic epithelium and
later differentiate into the neurons that will innervate the
hair cells . It has been shown in zebrafish that correct
neuroblast formation requires Notch-mediated lateral signal-
ing ; however, in mammals it has not been shown
definitively that this is the role that the Dll1 gene plays at
early stages. This leaves open the possibility that this early
domain of DLL1 expression may be at least partially involved
in prosensory specification, similar to the JAG1 expression
Nonsensory Defects in Jag1-cko Inner Ears
In addition to the defects in sensory formation in the Jag1-
cko inner ears, the mutant inner ears also exhibited non-
sensory defects. Specifically, the semicircular canals were
largely absent, with the exception of portions of the anterior
and lateral canals. In addition, all three ampullae were absent,
the utricle was small, and the cochlea was undercoiled. Based
on recent studies, it is likely that these defects are secondary
to the sensory defects. For example, it has been shown that
FGFs expressed in the sensory cristae promote semicircular
canal formation through up-regulation of BMP2 . Thus,
loss of the cristae would be expected to have a severe affect
on canal formation. Emerging evidence from mouse mutants
has demonstrated that genes involved in sensory formation
result in severely malformed inner ears. For example,
mutations in the Sox2 gene lead to malformations very
similar to those described here in Jag1-cko mutants. The inner
ears of embryos homozygous for two different mutant alleles
Figure 9. At E12.5 JAG1 Is Expressed within the Prosensory Domain and SOX2 Expression Is Down-Regulated within This Domain in Jag1-cko Cochleae
(A, B, D, and E) Alternate sections from a control embryo processed for immunocytochemistry using antibodies against either JAG1 or SOX2. Note the
similar domain occupied by both JAG1 and SOX2 in the base of the cochlea (A and B; brackets). In the apical region, the two proteins are not colocalized
(D and E). SOX2 is not expressed in the basal portions of the Jag1-cko cochlea (C) and shows only weak expression in the apex (F). bv, blood vessel; cd,
PLoS Genetics | www.plosgenetics.org January 2006 | Volume 2 | Issue 1 | e40035
JAG1 Function in Ear Sensory Development
of Sox2, Sox2lcc/lccand Sox2ysb/ysb, showed disrupted canal
formation; smaller utricular and saccular compartments;
and thinner, undercoiled cochleae . In addition, FGF10
mouse knockouts also showed disrupted cristae development
associated with loss of canal structures . However, unlike
the canal structures, cochlear formation does not appear to
be strictly dependent on development of the organ of Corti,
as a cochlea, albeit short and thin, will form in the absence of
any sensory formation . However, normal cochlear length
appears to be dependent on sensory formation, at least
partially through convergent extension. Recently, a number
of genes have been found in the cochlea that lead to defects
in planar cell polarity as well as a shortened cochlea,
presumably because of defects in convergent extension
[38,41]. Therefore it is likely that the shortened cochlea
observed in Jag1-cko mutants is at least partially a result of
failure of convergent extension caused by a reduction in the
number of sensory precursors.
The data presented here demonstrate that the Jag1 gene is
required for sensory precursor development in the inner ear.
Further studies are required to establish the exact role that
JAG1-mediated Notch signaling plays in early sensory
progenitors, and also its relationship to the roles played by
FGF signaling and SOX2 expression. Understanding how the
sensory precursors form is an important prerequisite for
regeneration studies that may provide molecular tools to
treat hearing loss and vestibular disorders .
Materials and Methods
Construction of the Jag1floxneoallele. To construct the Jag1floxneo
allele, bacterial artificial chromosome clones containing the Jag1
genomic locus were isolated from a RPCI-22 (129S6/SvEvTac) mouse
bacterial artificial chromosome library (filters obtained from Re-
search Genetics) by hybridization to a 1.8-kb mouse Jag1 cDNA probe.
To make the shorter 59 homology region of the targeting vector, a 2.2-
kb KpnI fragment upstream of exon 4 was isolated, blunt-ended, and
subcloned into the SmaI site of a modified pBS vector that contained
a loxP-FRT-PGKneo-FRT cassette. A 1.5-kb KpnI fragment that
contained exon 4 was also subcloned into the loxP-FRT-PGKneo-
FRT cassette. To construct the longer 39 homology region, a 3.5-kb
KpnI-SmaI fragment containing exon 5 was blunt-ended and
subcloned into the EcoRV site of another modified pBS vector that
contained a single loxP site. A 3.5-kb SmaI-SalI fragment from this
construct was then cloned into the SmaI-XhoI site of a pKO 905
vector containing a diphtheria toxin gene for negative selection. A
5.7-kb SalI-NotI fragment from the loxP-FRT-PGKneo-FRT construct
was then cloned into the SalI-NotI sites of the pKO 905 vector
containing the 39 homology region to generate the final Jag1floxneo
targeting vector (see Figure 1).
Generation of Jag1floxmice. The Jag1floxneotargeting construct was
linearized with Not1 and electroporated into CJ7 embryonic stem
(ES) cells, as described previously . DNA from 288 ES cell clones
was screened by PCR using an internal/external primer set, and
positive clones were then confirmed by Southern blot by probing
EcoRI-digested DNA with an external 1.7-kb Stu1-EcoRI fragment
located 39 to the targeting construct (see Figure 1). This probe also
detected partial recombination events in which the distal loxP site
was lost; in these cases a slightly larger fragment (11 kb rather than 9.3
kb) was obtained (see Figure 1A). The presence of the distal loxP site
was further confirmed by PCR using primers that flanked the loxP
site (DSLF and J1LoxR1; see below for sequences). Correctly targeted
clones were injected into C57BL/6J (B6) blastocysts, and chimeric
Figure 10. JAG1 and SOX2 Mark the Prosensory Regions of the Vestibule, and SOX2 Expression Correlates with Impaired Sensory Formation in the Jag1-
(A and B, D and E, G and H) Alternate sections demonstrating either JAG1 or SOX2 expression in the vestibular regions of control inner ears.
(C, F, I) Similar sections through the Jag1-cko inner ear demonstrating SOX2 expression. Dotted lines indicate regions where the cristae and ampullae
are missing in the Jag1-cko inner ear. ac, anterior cristae; lc, lateral cristae; pc, posterior cristae; sac, saccular macula; ut, utricular macula.
PLoS Genetics | www.plosgenetics.orgJanuary 2006 | Volume 2 | Issue 1 | e4 0036
JAG1 Function in Ear Sensory Development
mice were obtained. Chimeric male mice were mated to B6 females
and the agouti progeny were assayed for the presence of the
Jag1floxneoallele by PCR using Jag1floxneospecific primers. Jag1floxneo/þ
mice were intercrossed to create homozygous Jag1floxneo/Jag1floxneo
offspring. Homozygous Jag1floxneo/Jag1floxneomice appeared normal
and healthy, suggesting that the neomycin resistance cassette
(PGKneo) did not adversely affect Jag1 expression. To control for
any possible effects from the presence of the PGKneo cassette, the
FRT-flanked PGKneo cassette was deleted by mating Jag1floxneomice
to FLPe-recombinase expressing mice (Gt[ROSA]26Sor
Jackson Laboratory, Bar Harbor, Maine, United States) to produce
Jag1floxmice. Both Jag1floxneoand Jag1floxmice were used in these
experiments, and no differences in the resulting phenotype were
observed. To differentiate the deleted form of this allele from our
previously reported Jag1 null mutant allele (Jag1del1) , we
designate the Jag1 allele generated by Cre recombinase-mediated
deletion of the Jag1floxor Jag1floxneoalleles the Jag1del2allele.
Mouse husbandry and genotyping. Foxg1-Cre mice (; gift of Rob
Burgess) were maintained on an outbred Swiss Webster background.
ZP3-Cre mice (; gift of Mimi de Vries and Barbara Knowles) were
maintained on a B6 background. Typically, males that were hetero-
zygous for both a Foxg1-Cre allele and our previously constructed Jag1
to Jag1flox/Jag1floxfemales that were maintained on a B6/129 back-
ground. Mice of the genotypes Foxg1-Cre/þ; Jag1del1/Jag1floxand Foxg1-
Cre/þ; Jag1del1/Jag1floxneowere used interchangeably, and are designated
as Jag1-cko mice in this report.
To genotype the Jag1floxmice, the primers used were: DSLF, 59-
TCAGGCATGATAAACCCTAGC-39 (forward) and J1LoxR1, 59-CTA
CATACAGCATCTACATGC-39 (reverse); these primers flank the 59
LoxP site. To genotype for CRE-mediated recombination, a primer
upstream of the 39 LoxP site was used: J1FlpF1, 59-CAGGTT
GAGTGCTGACTTAG-39, along with the J1LoxR1 reverse primer.
To genotype for the Foxg1-Cre and ZP3-Cre alleles, Cre-specific primers
were used: Cre1, 59-TGATGAGGTTCGCAAGAACC-39 (forward) and
Cre2, 59-CCATGAGTGAACGAACCTGG-39 (reverse). Jag1del1primers
were as follows for the mutant: JGKO1, 59-TCTCACTCAGGCATGA
TAAACC-39 (forward) and SOL1, 59-TGGATGTGGAATGTGTGC
GAG-39 (reverse). A different reverse primer, JGKO2, 59-TAACGGG
GACTCCGG ACAGGG-39 was used to detect the wild-type allele.
Littermates (wild type, Jag1del1/þor Jag1flox/þ) were used as controls for
Paintfilling and scanning electron microscopy. The paintfilling of
the Jag1-cko inner ears was performed at E15.5. The technique was
performed as previously described . Inner ears were prepared for
SEM as described previously using a version of the osmium tetroxide-
thiocarbohydrazide method . Specimens were examined with a
Hitachi 3000N scanning electron microscope (Hitachi, Tokyo, Japan).
Immunohistochemistry and lectin staining. For immunohisto-
chemistry, embryonic heads were bisected and fixed for 1–2 h in
4% paraformaldehyde in PBS. Half heads were embedded in paraffin
wax, and immunocytochemistry was performed on standard 7-micron
sections. Antibodies used included anti-Myo7a (1:1,000; gift of A. EL-
Amraoui and C. Petit), anti-p27kip1(1:100; Neomarkers, Stratech,
Soham, Cambridgeshire, United Kingdom), anti-SOX2 (1:2,000;
Chemicon, Temecula, California, United States; AB5603), anti-JAG1
(1:100; Santa Cruz Biotechnology, Santa Cruz, California, United
States; H-114) and anti-S100A1 (1:50; Dako, Glostrup, Denmark). For
all antibodies used, an antigen retrieval step was performed by
boiling the sections for 10 min in 10 mM citric acid. Secondary
antibodies usedwereeitherAlexa-Fluor 488or 546goat anti-mouseor
rabbit (1:400; Invitrogen, Carlsbad, California, United States). Slides
were coverslipped in Vectashield HardSet Mounting Medium with
DAPI (Vector Laboratories, Burlingame, California, United States).
Lectin staining was performed using the Griffonia simplifonia I lectin
(Vector Laboratories) essentially as described .
In situ hybridization and RT-PCR. Since the Jag1del2mutant allele
creates an in-frame deletion of the DSL domain, an in situ probe
was designed for detection of the floxed region of the Jag1floxallele
by in situ hybridization. The probe was created by amplifying a 433-
bp product encompassing exon 4 (primers: J1-420F, 59-CGACCG
TAATCGCATCGTAC-39 and J1-853R, 59-ATGCACTTGTCGCAGTA
CAG-39) and subcloning the product into the PCR II vector
(Invitrogen). For whole-mount embryos in situ, embryos were fixed
overnight in 4% paraformaldehyde. In situ hybridization was
performed essentially as described . For cochlear in situ, inner
ears were dissected from the head and fixed overnight in 4%
paraformaldehyde. After washing in PBS, the bony shell and stria
were removed from the cochleae and the samples were dehydrated
in methanol. In situ hybridization was performed as described ,
with the exception of the posthybridization washes, which were
done according to . For RT-PCR, total RNA was extracted from
the E10.5 control and Jag1del2/Jag1del2embryos, using an RNAeasy kit
(Qiagen, Valencia, California, United States) and following the
manufacturer’s instructions. First-strand cDNA synthesis was per-
formed using the AMV reverse transcriptase (Promega, Madison,
Wisconsin, United States) with specific primer J1-961R (59-AGTCC
CACAGTAATTCAGATC-39). Products were amplified from cDNA
using primers that flanked exon 4 (J1-420F, 59-CGACCGTAATCG
CATCGTAC-39 and J1-961R).
We thank Drs. A. El-Amraoui, C. Petit, R. Burgess, M. de Vries, and B.
Knowles for reagents; Peter Finger of the Jackson Laboratory
Histopathology and Microscopy Services for help with the SEM
processing; and the Jackson Laboratory Cell Biology and Micro-
injection Core Facility for the blastocyst injections. This work was
supported by grants from the National Institutes of Health to AEK
(DC05865) and TG (NS036437 and DK066387), and from the Jackson
Author contributions. AEK and TG conceived and designed
experiments. AEK and JX performed experiments. All authors
analyzed the data. AEK wrote the paper.
Competing interests. The authors have declared that no competing
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PLoS Genetics | www.plosgenetics.org January 2006 | Volume 2 | Issue 1 | e40038
JAG1 Function in Ear Sensory Development