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THE ANATOMICAL RECORD 254:127–134 (1999)
Comparison of Ephrin-A Ligand
and EphA Receptor Distribution
in the Developing Inner Ear
LYNNE M. BIANCHI* AND HONG LIU
Department of Otolaryngology and Communicative Sciences, Medical University
of South Carolina, Charleston, South Carolina 29425
ABSTRACT
Members of the recently discovered Eph family appear to play important roles in a variety of developmental processes including tissue segmentation, cell migration and axonal guidance. To begin to understand the
functions of the EphA subclass of receptors and their corresponding GPIlinked (ephrin-A) ligands in the inner ear, a developmental immunohistochemical analysis was completed. The results indicated that the ligands
ephrin-A1 and ephrin-A2 were localized mainly at cellular boundaries in the
inner ear. Ephrin-A1 was detected mainly in the epithelial cells lining the
fluid filled ducts of the inner ear, whereas ephrin-A2 was prominently
expressed in connective tissue regions. The receptor EphA4 was detected in
vestibular hair cells. EphA5 and EphA7 were detected mainly in cochlear
and vestibular supporting cells. These results suggest that these Eph
molecules play a role in establishing the formation and cellular organization
of the complex inner ear labyrinth. Additionally, all of the ligands and
receptors evaluated were expressed in vestibular and cochlear neurons at
various developmental stages, suggesting they may play a role in establishing or maintaining innervation to the inner ear. Anat Rec 254:127–134,
1999. r 1999 Wiley-Liss, Inc.
Key words: Eph; immunohistochemistry; hair cell; gerbil; auditory
The developing inner ear forms initially as a simple
sac-like structure, the otocyst, that ultimately differentiates into the complex labyrinth comprising auditory (cochlear) and vestibular regions. Each of these regions is
innervated by the corresponding branch of the eight
cranial nerve. In order to develop from the simple, sac-like
otocyst, the inner ear undergoes a series of morphological
changes requiring cellular migration, reorganization and
transformation (reviewed by Fritzsch et al., 1998). The
cellular and molecular mechanisms underlying this complex development are only beginning to be defined.
Recently, a family of receptor tyrosine kinases (Ephs)
and their associated, membrane-attached ligands (ephrins) have been discovered. Together, these receptors and
ligands comprise the ‘‘Eph family.’’ The family is divided
into two subclasses based in part on preferential binding
interactions (Gale et al., 1996a). In one ligand subclass
(ephrin-A1–A5) the ligands are membrane attached by
glycosylphosphatidylinositol (GPI) linkage and bind primarily to receptors of the EphA subclass (EphA1–A8).
Ligands of the ephrin-B subclass (ephrin-B1–B3) are
membrane associated through a transmembrane domain.
r 1999 WILEY-LISS, INC.
These ligands bind mainly to the EphB subclass of receptors (EphB1–B6). The ligands and receptors appear to
interact through cell-to-cell contact (Davis et al., 1994).
Members of the Eph family play a role in the development and patterning of a variety of tissues including those
of the nervous system, limb bud, kidney and vascular
system (Nieto et al., 1992; Ruiz and Robertson, 1994; Ellis
et al., 1995; Gale et al., 1996a,b; Flenniken et al., 1996;
Kilpatrick et al., 1996; Daniel et al., 1996; Krull et al.,
1997; Wang et al., 1998). Eph ligands and receptors are
often localized in complementary patterns on adjacent
tissues, supporting the idea that ligands and receptors
interact by direct cell-cell contact (Gale et al., 1996a;
Flenniken et al., 1996). Eph molecules have also been
Grant sponsor: NIH/NIDCD; Grant number: DC03121.
*Correspondence to: Lynne M. Bianchi, Neuroscience Program,
Oberlin College, 130 West Lorain Street, Oberlin, OH 44074.
E-mail: lynne.bianchi@oberlin.edu
Received 22 July 1998; Accepted 10 September 1998
128
BIANCHI AND LIU
reported to influence actin polymerization to modify cellular morphology (Meima et al., 1997a,b).
Recent studies have reported the presence of several
Eph family members in the developing inner ear (Ciossek
et al., 1995; Lee et al., 1996; Henkemeyer et al., 1994;
Pickles and van Heuman, 1997; Bianchi and Gale, 1998)
and mature cochlea (Lee et al., 1996; Bianchi and Gale,
1998). In the present study, the immunohistochemical
localization of two ephrin-A ligands and three EphA receptors was examined in developing and mature vestibular
and cochlear regions of the inner ear using available
antisera. The distributions of these molecules suggest that
Eph ligands and receptors may play diverse roles in
boundary formation, tissue segmentation, pattern formation and target innervation in the inner ear.
MATERIALS AND METHODS
Tissue Collection
Tissues were harvested and processed for immunohistochemistry as described previously (Bianchi and Gale,
1998). Tissues were obtained from Mongolian gerbils
(Meriones unguiculatus) at embryonic day 14 (E14) through
adult stages. At least two animals were examined from
each stage evaluated (E14, E15, E18, postnatal day 1 [P1],
P3, P6, P8, P12, P14, 2 months, and 4 months). Embryonic
tissues were harvested from timed-pregnant gerbils euthanized by inhalation of carbon dioxide. Early postnatal
(P2–3) animals were sacrificed by inhalation of carbon
dioxide. Older animals (P4–adult) were heavily anesthetized with urethane, then exsanguinated by transcardial
perfusion with warm 0.9% saline solution followed by
fixative. All procedures were approved by the MUSC
Institutional Animal Care and Use Committee.
Tissues were fixed in either paraformaldehyde (4% in
0.1 M phosphate buffer, pH 7.2) or zinc formalin (10%
formalin in 0.9% saline containing 0.5% zinc dichromate,
pH 5.0). Embryonic and early postnatal animals were
bisected along the midsagittal plane and immersion fixed
for 6–18 hr. Older animals were perfused as described
above, the inner ears dissected, the stapes removed, the
round window pierced and 1 ml of fixative was infused
through the oval window. The inner ears were then
immersion fixed for an additional 30 min. Following
fixation, tissues were rinsed in PBS. Older tissues (P8–
adult) were decalcified for 24–72 hr in 0.12 M EDTA (pH
7.0). Tissues were then dehydrated through a graded
series of alcohols, cleared in histoclear (National Diagnostics) then embedded in Vision 52°C paraffin (Vision Medical).
Immunohistochemistry
Eph family members. Immunohistochemistry was
performed as described previously (Bianchi and Gale,
1998). Polyclonal antisera recognizing ephrin-A1 (B61),
ephrin-A2 (Elf-1), EphA4 (Hek 8), Eph A5 (Hek 7) and
EphA7 (Hek 11) were obtained from Santa Cruz Biotechnology. Two or three tissue sections were mounted on each
slide. Sections were deparaffinized, rehydrated and treated
with 0.3% hydrogen peroxide. Sections were blocked with
1% normal goat serum for 20 min, then incubated overnight in primary antisera at 4°C at optimal dilutions
determined by titration studies. Each antiserum was run
in duplicate in two to five separate trials for each developmental stage examined. Controls consisted of sections
incubated in normal serum lacking primary antisera or
preabsorption with a tenfold excess of an appropriate or
inappropriate peptide (Santa Cruz Biotechnology, Santa
Cruz, CA). Only those sections treated with antisera
preabsorbed with an inappropriate peptide showed immunoreactivity. The following day, sections were rinsed in
PBS, incubated in biotinylated goat anti-rabbit IgG for 20
min, rinsed in PBS, then incubated for 30 min with the
avidin-biotin horseradish peroxidase complex (ABC, Vector Labs, Burlingame, CA). Sites of binding were visualized by incubation in 3,38-diaminobenzidine-HCl (DAB)H2O2 substrate medium. Intensity of immunoreactivity
was scored subjectively (⫹⫹, ⫹, ⫹/⫺, ⫺) by examining
multiple sections prepared on different days.
Actin. To test cross-reactivity of actin and EphA7, both
EphA7 antisera and a smooth muscle ␥ actin monoclonal
antibody (HH35, DAKO, Glostrup, Denmark) were preincubated with tenfold and 100-fold excess of chicken gizzard
actin for 3 hr at room temperature. Following incubation,
tissues were processed as described above.
RESULTS
Table 1 lists the sites of immunoreactivity for ephrin-A1,
ephrin-A2, EphA4, EphA5 and EphA7 in the developing
and mature inner ear.
Distribution of Ephrin-A1 and Ephrin-A2
Ligands
Ephrin-A1 in epithelial cells lining the vestibular and cochlear labyrinth. Ephrin-A1 was faintly
detected in undifferentiated E14 and E15 vestibular epithelia. By E18, weak immunoreactivity was detected at the
base of the vestibular hair cells and prominent immunoreactivity was noted surrounding the epithelial cells lining
the vestibular wall (Fig. 1A). The location of these cells
suggests that they are the undifferentiated transitional
and dark cells that border the vestibular sensory epithelia.
By the first postnatal week, immunoreactivity was detected only as a thin line covering the epithelial cells that
line the non-sensory portion of the vestibular apparatus.
A similar pattern of ephrin-A1 immunostaining was
noted in the epithelial cells of Reissner’s membrane of the
cochlea. The developmental maturation of Reissner’s membrane is clearly seen in the three turns of the E18 cochlea
(Fig. 1B). In the apical turn where Reissner’s membrane
had not yet separated the scala vestibuli from the scala
media, ephrin-A1 was located in epithelial cells surrounding the upper portion of the cochlear duct (Fig. 1B). In the
more mature middle turn, where there was mesenchymal
thinning in the area forming scala vestibuli, ephrin-A1
immunoreactivity was detected in the epithelial cells of
Reissner’s membrane. Immunoreactivity was also detected on the lateral side of the cochlear duct where the
outer sulcus forms (Fig. 1B). In the basal turn, where the
scala vestibuli was more developed, ephrin-A1 immunoreactivity was predominantly expressed surrounding the
epithelial cells of Reissner’s membrane (Fig. 1B, C). The
thickness of the region of immunoreactivity along Reissner’s membrane decreased by P2, coinciding with the
developmental reduction in thickness of Reissner’s membrane. In the adult, ephrin-A1 was detected only as a thin
line on the scala media side of Reissner’s membrane in all
turns. Thus, ephrin-A1 expression became more restricted
as the epithelial cells lining the fluid filled cavities of the
129
EPH MOLECULES IN THE INNER EAR
TABLE 1. Relative intensity of immunoreactivity for ephrin-A1, ephrin-A2, ephA4,
ephA5, and ephA7 in developing and mature inner ear regions
Neurons
ephrin-A1
Embryonic
Postnatal
Adult
ephrin-A2
Embryonic
Postnatal
Adult
EphA4
Embryonic
Postnatal
Adult
EphA5
Embryonic
Postnatal
Adult
EphA7
Embryonic
Postnatal
Adult
Undifferentiated
cells
Sensory
hair cells
Support
cells
⫹/⫺
⫹
⫹⫹
⫹
n/d
n/d
⫹/⫺ (V)
—
—
⫹⫹
⫹
⫹/⫺
—
—
⫹ (V)
⫹/⫺ (C)
⫹/⫺
n/d
n/d
—
—
—
⫹⫹ (V)/⫹ (C)
⫹⫹ (V, C)
⫹ (V, C)
⫹
⫹⫹
⫹⫹
⫹/⫺
n/d
n/d
⫹ (C)
⫹⫹ (C, V)
⫹⫹ (C)
⫹/⫺ (C)
⫹⫹ (C)
⫹⫹ (C)
⫹ (V)
⫹ (C & V)
⫹ (C & V)
⫹
n/d
n/d
⫹/⫺
⫹/⫺
—
⫹ (C)
⫹⫹ (C)
⫹⫹ (C)
⫹/⫺ (C)
⫹/⫺ (C)
⫹/⫺ (C)
⫹/⫺
n/d
n/d
⫹/⫺
⫹
⫹⫹
⫹
⫹⫹
⫹⫹
⫹⫹, intense immunoreactivity; ⫹, positive immunoreactivity; ⫹/⫺, weak immunoreactivity; ⫺, no
immunoreactivity; n/d, not determined. All regions were positive in both vestibular (V) and cochlear
(C) regions, unless otherwise noted. Immunostaining results for cochlear regions treated with
ephrin-A2 and EphA4 antisera were shown in Bianchi and Gale (1998).
vestibular and cochlear labyrinths attained their adult
form. None of the EphA receptors examined in the present
study were in cells adjacent to ephrin-A1 expression. Thus,
another member of the EphA subclass may interact with
the ephrin-A1 to mediate maturation of these cells.
Expression of Ephrin-A2 in connective tissue
regions. In the embryonic vestibule, ephrin-A2 was detected along the lateral regions of the crista ampullaris
(Fig. 2A). Ephrin-A2 was localized primarily below the
transitional cells adjacent to the sensory epithelium. By
P2, immunoreactivity in the crista extended below both
transitional and dark cells, as well as in a region directly
below the central most sensory epithelium. During the
second postnatal week, immunoreactivity continued to be
expressed in these regions and began to thicken and
become more diffuse (Fig. 2B). This pattern of immunostaining persisted into adult stages (2–4 months), although the intensity of staining appeared to decrease
slightly.
Ephrin-A2 was also located below the sensory epithelium of the macula from embryonic through adult stages.
Through the first postnatal week, immunoreactivity was
limited to a discrete region below the sensory epithelium
(Fig. 2C). Similar to the staining observed in the crista, the
region of immunostaining below the macular sensory
epithelium gradually became thicker and more diffuse
beginning in the second postnatal week (Fig. 2D). Immunoreactivity persisted in the adult macula. Thus, in both the
crista and macula, ephrin-A2 immunoreactivity was highly
expressed in connective tissue regions adjacent to or below
vestibular sensory epithelium. Our previous studies in the
cochlea also found ephrin-A2 immunoreactivity in connective tissues adjacent to and below cochlear sensory epithelium (Bianchi and Gale, 1998).
Expression of the EphA4, EphA5, and EphA7
Receptors
EphA4 expression in sensory hair cells. EphA4
was highly expressed in vestibular hair cells of both the
crista and the macula beginning at early postnatal stages
(P2–P8; Fig. 3). Immunoreactivity in hair cells continued
through the second postnatal week, but was not observed
in adult hair cells. It appeared that both flask-shaped type
I and cylindrical type II hair cells were immunopositive for
EphA4. EphA4 was previously found in interdental and
sensory hair cells of the developing cochlea (Bianchi and
Gale, 1998).
EphA5 primarily limited to cochlear regions.
EphA5 was barely detectable in the vestibular epithelium
from embryonic (E15) through early postnatal stages (P3),
and was absent in adult vestibular epithelia. In the
postnatal (P1–P14) and adult cochlea, EphA5 was detected
in fibrocytes of the lateral wall and in interdental cells of
the spiral limbus (not shown). These areas of immunoreactivity in the cochlea were similar to the pattern previously
described for EphA4 (Bianchi and Gale, 1998). It appears
that EphA4 and EphA5 overlap in some cochlear regions,
but not in vestibular regions.
Co-localization of EphA7 and actin. EphA7 was
detected as a very thin band along the apex of the sensory
hair cells in embryonic and postnatal crista and macula
(E18–P24). In the adult, some fibrocytes below the macula
also appeared immunopositive (not shown). Other regions
of the vestibular system, including vestibular neurons,
were not immunoreactive.
In the cochlea, EphA7 was detected in the basal cells of
the stria vascularis (Fig. 4A). Immunoreactivity was also
detected in the pillar cells and in the upper portion of
130
BIANCHI AND LIU
Deiters’ cells (Fig. 4B), in an area termed the Rosette
complex (Spicer and Schulte, 1993).
The regions of EphA7 immunoreactivity in the cochlea
were remarkably similar to those previously described for
smooth muscle ␥ actin (Nakazawa et al., 1996). To test
whether the EphA7 antiserum was detecting actin, EphA7
and a ␥ actin monoclonal antibody (HH35, DAKO Corporation) were preincubated with tenfold and 100-fold excess of
actin from chicken gizzard. Preabsorption eliminated the
staining observed with the anti-actin antibody. In contrast,
EphA7 immunoreactivity was unaffected at either concentration of actin (not shown). Thus, EphA7 appears to
co-localize to regions expressing ␥ actin.
Distribution of Ephrin-A Ligands and EphA
Receptors in Vestibular and Cochlear Neurons
Neuronal Expression. Table 1 summarizes the expression of Ephrin-A and EphA molecules in developing
and adult vestibular and cochlear neurons. Vestibular
neurons were immunoreactive for ephrin-A1 from embryonic through adult stages. The intensity of immunoreactivity increased from early postnatal through adult stages,
where it reached its maximum. Similarly, cochlear neurons showed only faint immunoreactivity at embryonic
stages, but increased immunoreactivity at early postnatal
and adult stages. At embryonic stages, more vestibular
neurons were positively stained than cochlear neurons.
Postnatally (⬃P2–10), it appeared that cochlear neurons
in the basal turn were more intensely stained than those
in the apical turn. Ephrin-A2 was not detected in embryonic or early postnatal vestibular or cochlear neurons, but
was detected in adult vestibular and cochlear neurons.
Ephrin-A2 immunoreactivity of adult vestibular neurons
appeared more intense than that observed in adult cochlear neurons.
Vestibular neurons were immunopositive for EphA4
from embryonic through adult stages, similar to the staining previously observed in cochlear neurons (Bianchi and
Gale, 1998). EphA5 was detected in embryonic, postnatal
and adult vestibular neurons. In contrast, cochlear neurons were not as immunoreactive as vestibular neurons
until P2, suggesting that expression of EphA5 occurred
first in vestibular, then in cochlear neurons. Cochlear
neurons were only faintly immunoreactive for EphA7 at
embryonic through adult stages. Vestibular neurons did
not appear to be immunoreactive for EphA7.
DISCUSSION
Fig. 1. Ephrin-A1 immunoreactivity in epithelial cells at E18. A:
Ephrin-A1 was localized to the epithelial cells of the vestibular wall
(arrow). B, C: Ephrin-A1 was detected in epithelial cells of the developing
cochlear duct, particularly in those forming Reissner’s membrane (arrows,
C). sv, scala vestibuli; sm, scala media. Magnification: A, 200⫻; B, 133⫻;
C, 328⫻.
The Eph family of receptors and corresponding membrane-bound ligands has been found to play a variety of
roles in tissue segmentation, axonal outgrowth and boundary formation (Henkemeyer et al., 1994; Drescher et al.,
1995; Cheng et al., 1995; Ruiz and Robertson, 1994; Gale et
al., 1996a,b; Daniel et al., 1996; Flenniken et al., 1996;
Winning et al., 1996; Krull et al., 1997; Wang and Anderson, 1997). The mammalian inner ear is a structure that
develops from a simple, epithelial sac to a highly complex
labyrinth comprising five vestibular organs (the utricular
and saccular maculae and the three cristae ampullares)
that regulate balance, and the coiled, snail-shaped cochlea
that regulates hearing. Several studies have reported the
expression of various Eph molecules in the early embryonic inner ear (Ciossek et al., 1995; Lee et al., 1996;
Henkemeyer et al., 1994; Pickles and van Heuman, 1997;
EPH MOLECULES IN THE INNER EAR
Fig. 2. Ephrin-A2 immunoreactivity in connective tissue regions. A, B:
Ephrin-A2 was localized to connective tissue regions adjacent to the
sensory epithelium of the crista. C, D: Ephrin-A2 was detected below the
sensory epithelium of the macula. Compared to earlier stages (E15, A; P6,
131
C), the thickness of immunoreactivity increased during the second
postnatal week (P14, B,D) in both regions. Insets (a–d) show enlargements of immunopositive areas indicated by arrows in A–D. Magnification:
A, B 219⫻, insets, 297⫻; C, D, 219⫻, insets, 281⫻.
132
BIANCHI AND LIU
Fig. 3. EphA4 immunoreactivity in vestibular hair cells of the P2
macula. Magnification: 266⫻.
Bianchi and Gale, 1998). However, the role of the Eph
family in inner ear development is still unknown. To begin
to understand possible functions of ephrin-A (GPI-linked)
ligands and their corresponding EphA receptors in the
inner ear, the present study consisted of a developmental
immunohistochemical analysis using currently available
polyclonal antisera against synthetic peptides (Santa Cruz
Biotechnology). In previous experiments, we compared
immunostaining using ephrin-A2, EphA4, ephrin-B1 and
EphB1 antisera to staining obtained with Fc-tagged Eph
ligands and receptors (Bianchi and Gale, 1998). The
results indicated that both the antisera and Fc-tagged
molecules revealed similar staining patterns, supporting
the idea that the antisera were detecting Eph molecules.
However, the data did not rule out the possibility that the
antisera recognized more than one member of the Eph
family. The staining patterns in that study, as well as the
present study, indicate that each antiserum is immunoreactive at different sites within the inner ear, suggesting
some degree of specificity. However, because the antisera
have not been thoroughly characterized, we cannot exclude the possibility that a single antiserum recognizes
multiple Eph family members.
In the present study, the expression patterns of ephrinA1, ephrin-A2, EphA4, EphA5 and EphA7 were compared
in the vestibular and cochlear regions of the inner ear.
These results and those of our previous study (Bianchi and
Gale, 1998), indicated that several Eph molecules are
localized at tissue boundaries in the inner ear. For example, at embryonic stages, ephrin-A1 and ephrin-A2
were predominantly expressed in cells lining the fluid
filled ducts of the inner ear and in connective tissue
regions adjacent to sensory epithelia, respectively. The
positions of these ligands suggests they may provide cues
to direct the migration of developing inner ear cells as they
form of the complex inner ear labyrinth. Thus, Eph
molecules may be necessary for establishing appropriate
cellular patterns in the inner ear.
Many of the Eph ligands and receptors were expressed
at borders between epithelial and mesoderm cells. For
example, ephrin-A1 was detected in epithelial cells lining
the fluid-filled spaces of the vestibular labyrinth and
cochlear duct. The function of ephrin-A1 in these regions
remains unclear. One possibility is that these ligands help
shape formation of the labyrinth by regulating epithelialmesenchymal interactions. Alternatively, ephrin-A1 may
be important for linking cells that are connected by tight
junctions to form barriers that limit the diffusion of ions.
In the embryonic cochlea, as the cochlear ducts began to
divide into the three separate fluid-filled chambers, expres-
sion of ephrin-A1 was located lining the scala media,
particularly in the epithelial cells of Reissner’s membrane.
Reissner’s membrane comprises epithelial cells facing
scala media, mesothelial cells facing scala vestibuli, and a
basement membrane separating the epithelial and mesothelial layers (Felix et al., 1993). Ephrin-A1 immunoreactivity was limited to the epithelial cells facing the scala
media. The mesothelial cells on the scala vestibuli side of
Reissner’s membrane were not immunoreactive. The expression of ephrin-A1 became more restricted by early
postnatal stages as the thicker, immature membrane
attained its thinner adult form. Ephrin-A1 has been shown
to function as an inhibitory molecule in other systems
(Nakamoto et al., 1996; Gao et al., 1996; Krull et al., 1997),
and therefore, may inhibit the migration of mesothelial
(mesenchymal) cells from the scala vestibuli side of Reissner’s membrane. Thus, ephrin-A1 may provide a signal
that assists in the formation of the boundary between
scala media and scala vestibuli.
The epithelial cells of Reissner’s membrane are connected by tight junctions to form a barrier that prevents
the intermixing of the endolymph (high potassium, low
sodium) of the scala media with the perilymph (high
sodium, low potassium) of the scala vestibuli. Similarly,
ephrin-A1 immunoreactivity was also detected on the
endolymph side of the vestibular wall around immature
transitional and dark cells. Adjacent to these epithelial
cells lies connective tissue, which in turn borders perilymph. Thus, ephrin-A1 was detected in cochlear and
vestibular epithelial cells that are tightly joined to separate regions of endolymph and perilymph.
None of the receptors analyzed in the present study were
expressed in regions complementary to the epithelial cells
positive for ephrin-A1. Thus, it is not yet known which
receptors are likely to interact with ephrin-A1 in these
regions. Availability of additional EphA antisera will be
necessary to address this issue and to provide further
insight into the functional significance of ephrin-A1 in
epithelial cells lining inner ear compartments containing
endolymph.
In the macular regions, another GPI-linked ligand,
ephrin-A2, was expressed in the connective tissue directly
below the epithelia. In the cristae, ephrin-A2 was localized
to regions below the transitional and dark cells and a small
region below the central-most hair cells. The embryonic
immunostaining patterns could indicate a role for ephrin-A2 in establishing epithelial boundaries. During postnatal stages the thickness of the immunostaining increased and ephrin-A2 continued to be expressed in these
regions through adult stages. It is unclear what function
ephrin-A2 may play in the adult vestibule. This increased
thickness of immunostaining in vestibular regions is in
contrast to the ephrin-A2 immunostaining in the spiral
limbus and inner sulcus of the cochlea. In the postnatal
spiral limbus and inner sulcus, ephrin-A2 immunoreactivity became more restricted from P2 to P6 (Bianchi and
Gale, 1998), coinciding with maturation of these regions.
In the vestibular system, EphA4 was expressed mainly
in vestibular hair cells in the upper regions of the epithelium. Occasional, scattered supporting cells located above
the basement membrane appeared immunopositive. EphA4
hair cell expression appeared to be distributed throughout
the epithelia and did not appear limited to type I or type II
hair cells. Immunoreactivity was detected from early
postnatal stages through the second postnatal week. By
EPH MOLECULES IN THE INNER EAR
133
Fig. 4. EphA7 immunoreactivity in the adult organ of Corti. A: EphA7 was localized to the basal cells of the
stria vascularis. B: Pillar cells and Deiters’ cells (arrows) were also immunopositive for EphA7. Magnification:
A, 228⫻; B, 203⫻.
adulthood, EphA4 immunoreactivity was absent in vestibular hair cells.
During development, the hair cells of the cristae mature
from the apex to the sides. Similarly, hair cells of the
maculae develop from a central to peripheral direction
(Sans and Chat, 1982). Nerve fiber innervation appears to
follow this same temporospatial pattern. The developing
vestibular epithelium is initially thicker than in the adult
form. The thinner adult morphology is obtained as the
supporting cells progressively rearrange into a monolayer
below the sensory hair cells. The onset of EphA4 expression occurred after the period hair cell terminal mitosis
and epithelial reorganization. However, final maturation
of the vestibular hair cells occurs during the first four
postnatal weeks in the rodent (i.e., mouse; Nordemar,
1983). The EphA4 receptors present on hair cells during
the first two postnatal weeks may interact with one of the
ephrin-A ligands to regulate final differentiation of vestibular epithelia.
EphA7 was not prominently expressed in vestibular
tissues, but was highly expressed in Deiters’ cells and
strial basal cells of the cochlea. These cells have been
proposed to be involved in ionic transport and regulation of
cochlear fluid homeostasis (Nakazawa et al., 1996). It is
unclear what role EphA7 plays in these cells. Similar to
epithelial cells of Reissner’s membrane, strial basal cells,
which are joined by tight junctions, are known to form a
diffusion barrier between perilymph and endolymph along
the lateral wall of the cochlea (Nakazawa et al., 1996). Eph
molecules may be involved in forming these cellular barriers. Basal cells are derived from mesoderm, again suggesting that Eph receptor-ligand interactions may be necessary for forming boundaries between epithelial and
mesoderm tissue.
It is interesting to note the co-localization of EphA7 with
smooth muscle ␥ actin in the Deiters’ cells and strial basal
cells of the cochlea. It has been proposed that actin in these
cells may be necessary for contractile processes regulating
ion homeostasis or micromechanical response properties of
the organ of Corti (Nakazawa et al., 1995, 1996). Previous
studies have noted a possible interaction of Eph molecules
with cytoskeletal elements. In neurons, it is thought that
Eph ligands and receptors may interact to regulate the
polymerization of filamentous actin. For example, ephrin-A5 appears to regulate growth cone collapse by disrupting the actin cytoskeleton (Meima et al., 1997a,b). Because
the function of actin in strial basal cells and Deiters’ cells is
not fully understood, it is difficult to speculate how Eph
molecules may interact to regulate actin-containing cells
in the inner ear. The co-localization may be merely coincidental, or may indicate a common link between Eph
molecules and cytoskeletal elements.
Vestibular and cochlear neurons were positive for both
ephrin-A ligands and EphA receptors. Several of these
molecules showed developmental increases in immunoreactivity. The expression in neurons could indicate that the
neurons interact with corresponding Eph ligands or receptors in the sensory epithelia to direct axonal growth to
sensory hair cells. For example, ephrin-A1 was detected at
the base of vestibular hair cells at E18. This ligand could
provide an inhibitory or attractive cue for vestibular
neurons expressing EphA receptors, such as Eph A4 or
EphA5. Additionally, the Eph molecules may be expressed
in central targets to guide central process to appropriate
regions in vestibular and cochlear nuclei, similar to roles
in other regions of the central nervous system (Drescher et
al., 1995; Cheng et al., 1995; Gao et al., 1996). Embryonically, EphA5 immunoreactivity appeared in vestibular
neurons prior to cochlear neurons. Vestibular neurons
precede cochlear neurons in terminal mitosis (Ruben,
1967; D’Amico-Martel, 1982), and thus, the differences in
timing of EphA5 immunoreactivity may coincide with
134
BIANCHI AND LIU
changes in the maturation of vestibular and cochlear
neurons. Postnatally, ephrin-A1 appeared to become more
highly expressed in the basal cochlear turns prior to the
less mature middle and apical turns. This gradient of
expression may be correlated with maturation of cochlear
neurons.
The results of the present study indicate several distinct
patterns of ephrin-A and EphA expression in the developing vestibular and cochlear labyrinth. Together, these
patterns suggest Eph molecules may mediate formation of
the inner ear and regulate epithelial-mesenchymal interactions at tissue boundaries. The patterns also suggest that
Eph molecules may be involved in regulating nerve fiber
innervation. Future studies examining in vitro inner ear
preparations, and the in vivo analysis of mice lacking one
or more of these molecules, will help elucidate the functions of these molecules in inner ear development.
ACKNOWLEDGMENTS
The authors thank B.H. Schmiedt for expert technical
assistance and Dr. B.A. Schulte for helpful comments on
the manuscript.
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