1 Department of Molecular and Human Genetics, Baylor College of Medicine,
Houston, TX 77030, USA
2 Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX
77030, USA
3 Division of Hematology/Oncology, Howard Hughes Medical Institute, Children's
Hospital and the Dana Farber Cancer Institute, Harvard Medical School, Boston,
MA 02115, USA
4 Program in Developmental Biology, Baylor College of Medicine, Houston, TX
77030, USA
5 Institute for Cellular Therapeutics and Department of Surgery, University of
Louisville, Louisville, KY 40202, USA
6 Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030,
USA
* Author for correspondence (e-mail: hbellen{at}bcm.tmc.edu)
Accepted 4 October 2002
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SUMMARY |
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Key words: Gfi1, Gfi1b, Senseless, PAG-3, Inner ear hair cell, Basic helix-loop-helix (bHLH), Deafness, Mouse
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INTRODUCTION |
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Recent publications suggest a variety of in vivo functions for
Gfi1 and Gfi1b. High levels of Gfi1 transgene result in a
block of T-cell lymphopoiesis (Schmidt et
al., 1998a; Schmidt et al.,
1998b
). Constitutive Gfi1 expression accelerates entry of
resting T cells into S phase of the cell cycle
(Karsunky et al., 2002a
); and
causes decreased levels of apoptosis, increased levels of cell proliferation
and a decrease in the levels of negative cell cycle regulators
p27KIP1 and pRb. Gfi1 may also regulate apoptosis through
repression of multiple pro-apoptotic regulators
(Grimes et al., 1996b
).
Outside the lymphoid system, Gfi1 is expressed in granulocytes and
activated macrophages (H. Hock, M. J. Hamblen, H. M. Rooke, D. Traver, R. T.
Bronson, S. Cameron and S. H. Orkin, unpublished)
(Karsunky et al., 2002b
).
Loss-of-function studies in mice mutant for Gfi1 indicate that it is
necessary during hematopoesis as it is required for the proper specification
and differentiation of neutrophils and macrophages (H. Hock, M. J. Hamblen, H.
M. Rooke, D. Traver, R. T. Bronson, S. Cameron and S. H. Orkin, unpublished;
Karsunky et al., 2002b
).
Overexpression of Gfi1b results in inhibition of G1 arrest and
differentiation by directly binding the p21WAF1 promoter
and repressing its activity (Tong et al.,
1998
). Gfi1b can also directly repress the activity of
tumor suppressor genes Socs1 and Socs3 by binding their
promoters (Jegalian and Wu,
2002
). The Gfi1b zinc-finger domain may also act as a
transcriptional activation domain (Osawa
et al., 2002
). Thus, Gfi1b may modulate transcription as a
repressor or activator depending on promoter and cell type context. Loss of
function studies in the mouse indicate that Gfi1b function is
required for hematopoiesis as it is required for erythroid and megakaryocytic
lineages. Mice deficient for Gfi1b are embryonic lethal and die by
E15.5 because of a complete lack of erythrocytes
(Saleque et al., 2002
).
The Gfi proteins have invertebrate homologues, including senseless
in Drosophila (Frankfort et al.,
2001; Nolo et al.,
2000
; Nolo et al.,
2001
) and pag-3 in C. elegans
(Cameron et al., 2002
;
Jia et al., 1996
;
Jia et al., 1997
). In
Drosophila, senseless is required during the development of the
embryonic and adult peripheral nervous system (PNS). Embryos that lack
senseless differentiate the majority of PNS cells, but most cells die
through apoptosis (Nolo et al.,
2000
; Salzberg et al.,
1997
). However, in adult sensory organs, senseless is
both necessary and sufficient for their development. Mosaic analysis in
imaginal discs shows that senseless mutant clones lack sensory
organs. Expression of senseless is dependent on the proper expression
of proneural genes, such as atonal, scute, achaete and
daughterless. senseless in turn is required for the upregulation and
maintenance of expression of the proneural genes in the sensory organ
precursors (SOP), as loss-of-function mutations in senseless abolish
the further upregulation and maintenance of proneural gene expression in the
SOPs. Ectopic expression of senseless induces ectopic proneural gene
expression and ectopic PNS organs. In addition, senseless has been
shown to synergize with the proneural genes
(Nolo et al., 2000
).
PAG-3 is a C. elegans homolog of Senseless and Gfi1.
pag-3 is involved in touch neuron gene expression and coordinated
movement (Jia et al., 1996;
Jia et al., 1997
). In
addition, null mutations of pag-3 can result in abnormal patterns of
apoptosis in the ventral nerve cord as well as abnormal differentiation of
certain interneurons and motoneurons. Hence, pag-3 functions in
diverse contexts within the developing nervous system. The finding that
pag-3 is expressed in many neuronal subtypes at different points in
development suggests that it cooperates with different factors to regulate the
expression of cell type- and developmental stage-specific sets of genes to
generate the complex pattern of neuronal subtypes seen in C. elegans
(Cameron et al., 2002
).
The above data suggest that this small but specific gene family of Zn-finger transcription factors plays similar as well as different roles in different species and different tissues. These include suppression of apoptosis, suppression of cell cycle checkpoints, as well as promoting cell fate determination and cell differentiation. However, in some lineages it is not obvious what the precise role of these proteins is.
Inner ear hair cell development
Mammalian inner ear hair cells function as mechanoreceptors to transduce
sound and proprioreception. The morphology and development of the mammalian
inner ear are very complex in nature. The major structures of the internal ear
consist of the utricle and saccule, three semicircular canals, the cochlea,
and the endolymphatic duct and sac. The sensory neuroepithelia are innervated
by the eighth cranial nerve which consists of two parts, the vestibular and
cochlear nerves. Vestibular hair cells are located in the macula of the
saccule and the utricle, as well as in the cristae located in the semicircular
canals. These hair cells fall into two types, Type I and Type II, and are
innervated by the vestibular nerve. The hair cells of the vestibule detect
linear acceleration and head position with respect to gravity. They are
responsible for the sense of balance and proprioreception. The auditory hair
cells in the organ of Corti located in the cochlea also fall into two
categories, inner hair cells and outer hair cells. They are innervated by the
cochlear nerve. These hair cells are responsible for auditory sensation. The
membranous labyrinth of the inner ear first begins to form from the otic cyst
and is visible in mice at E10.75. By E17.5 the gross anatomy of the inner ear
is mature (Cantos et al.,
2000). Many hearing impairments are caused by loss of sensory
neurons and inner ear hair cells (for a review, see
Petit et al., 2001
). Hence, a
better understanding of the genetic mechanisms responsible for the development
of these structures may help us dissect the mechanisms implicated in hearing
impairment or deafness.
A homology between hair cells in vertebrates and chordotonal organs in
flies has been recently revealed (Hassan
and Bellen, 2000). The bHLH proneural gene atonal was
shown to be required for the specification of chordotonal SOPs
(Jarman et al., 1993
). These
chordonal organs function as proprioreceptive organs and hearing devices
(Eberl, 1999
;
McIver, 1985
;
van Staaden and Römer,
1998
), much like the hair cells of the balance organs and the
auditory system. As mentioned previously, Atonal is required for
senseless expression in the SOP, and ectopic senseless
expression induces atonal expression (H. J. B., unpublished). One of
the mouse homologues of atonal, Math1 (Atoh1 Mouse
Genome Informatics) is expressed in the inner ear hair cells during
development. Math1-null mice die shortly after birth and lack hair
cells in balance organs and cochleae
(Bermingham et al., 1999
).
Interestingly, all the defects associated with loss of Math1 can be
rescued by the fly ato gene, suggesting that they are orthologs
(Wang et al., 2002
). In
addition, Math1 overexpression has been shown to induce hair cell
growth in inner ear epithelia (Zheng and
Gao, 2000
). Hence, Math1, like ato, is necessary
and sufficient for hair cell development in vertebrates.
Given the similarities between Math1 and atonal and the role of senseless in PNS development, we investigated the expression pattern and role of the senseless homolog, Gfi1, in hair cell development. We find that Gfi1 is expressed in many neuronal precursors as well as differentiating neurons during embryonic development. Consistent with these expression patterns, analysis of Gfi1 function in inner ear development in a previously generated Gfi1-mutant line (H. Hock, M. J. Hamblen, H. M. Rooke, D. Traver, R. T. Bronson, S. Cameron and S. H. Orkin, unpublished) revealed that mutant hair cells are initially specified and express many hair cell markers, including Math1. However, Gfi1 is required for proper differentiation and maintenance of inner ear hair cells. In Gfi1 mutant mice, the vestibular and cochlear hair cells are morphologically abnormal, hair cell organization within the sensory epithelia is aberrant, the outer hair cells in the organ of Corti express a neuronal marker, and cochlear hair cells degenerate after separation. Thus, Gfi1 is expressed in the developing nervous system and is required for the differentiation and survival of inner ear hair cells.
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MATERIALS AND METHODS |
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Embryo staging and tissue preparation
Embryos were considered to be E0.5 days on the morning the vaginal plug was
observed. To harvest the embryos, pregnant females were sacrificed by cervical
dislocation and the embryos dissected out of the uterus. Regions of the yolk
sac or tail were saved for genotyping. Embryos were fixed overnight in 4%
paraformaldehyde, dehydrated in an ethanol series and embedded in paraffin wax
for sectioning according to standard histological protocols. Sections (10
µm) were collected and analyzed by in situ hybridization or
immunohistochemistry. Ear tissue for postnatal stages was collected by
harvesting the temporal bones of the appropriately aged pup, fixing overnight
in 4% paraformaldehyde, decalcifying in 1.35 N hydrochloric acid for at least
an hour, dehydrating in an ethanol series and embedding in paraffin for
sectioning. Sections (10 µm) were collected and analyzed by
immunohistochemistry.
In situ analysis of Gfi1, Math1 and Brn3c
The cDNA probe for Brn3c (Pou4f3 Mouse Genome
Informatics) was kindly provided by Bill Klein. Probes for each of the genes
were transcribed in the antisense direction and labeled with digoxigenin using
the Dig RNA Labeling Kit from Roche. Probes were hybridized to paraffin
sections and detected by anti-digoxigenin antibody coupled to alkaline
phosphatase. Hybridization and stringent posthybridization wash steps were
performed at 65°C.
Immunohistochemistry
Anti-Myosin VI/VIIa was kindly provided by Tama Hasson. Anti-TUJ1 was
obtained from Babco. Anti-activated Caspase 3 was obtained from R&D
Systems. An anti-Gfi1 antibody was generated in guinea pig. This antibody was
raised against the domain of Gfi1 between the SNAG domain and the zinc fingers
(amino acids 20-256), cloned into pET28a. This domain does not display
homology to Gfi1b or other proteins. It is a specific nuclear antigen that is
not present in Gfi1 mutant mice and recognizes a specific band of the
appropriate molecular weight on western blots of Gfi1-expressing yeast
extracts and of mouse thymus protein extracts (data not shown). We used
antibodies to Myosin, TUJ1 and Caspase 3 at a 1:1000 dilution, and Gfi1 at a
1:2000 dilution, and followed the ABC Vectastain directions with secondary
anti-rabbit antibody (Myosin VI/VIIa and Activated Caspase 3), anti-mouse
antibody (TUJ-1) or anti-guinea pig antibody (Gfi1) followed by DAB staining.
Briefly, paraffin wax embedded sections were blocked in 1%
H2O2 in methanol for 20 minutes at room temperature,
rehydrated in a series of ethanols, boiled in citrate antigen retrieval
solution in a microwave for 5-10 minutes, and blocked with horse serum
(Vectastain) in PBS for 30 minutes at room temperature. Primary antibody was
diluted in blocking solution and incubated on the section overnight at
4°C. Slides were rinsed in PBS and incubated in secondary for 30 minutes
at room temperature. The slides were rinsed in PBS and incubated in Vectastain
ABC solution for 30 minutes. The slides were again rinsed in PBS and the
signal was detected with 2 mg/ml DAB, 0.02% H2O2 in PBS.
Some slides were counterstained with Hematoxylin.
ß-Gal staining
Mice heterozygous for the ß-galactosidase cassette in the place of
Math1-coding region were bred to Gfi1 heterozygous mice to
generate double heterozygotes, which in turn were crossed to obtain genotypes
that were Math1 heterozygous and either Gfi1 wild type or
Gfi1 null. This allowed us to visualize hair cells in the
Gfi1 null mutants by staining the tissues for ß-galactosidase.
Appropriately staged mice were harvested, fixed briefly in 4% paraformaldehyde
and stained overnight at 37°C for ß-galactosidase according to
established procedures (Ben-Arie et al.,
2000). The tissue was then fixed overnight in 4% paraformaldehyde,
and processed for paraffin wax embedding and sectioning or imaged immediately
for whole mounts.
TEM
Staged cochleae were dissected and fixed in 0.1 M cacodylate buffer, 1%
glutaraldehyde and 4% formaldehyde at 4°C for 2 hours. Specimens were then
rinsed in 0.1 M cacodylate buffer and post fixed in 1% osmium tetroxide in
cacodylate buffer at 4°C overnight. Samples were again washed in
cacodylate buffer and rinsed with distilled water. Specimens were stained with
4% uranyl acetate for 3 hours and again washed in distilled water. Specimens
were then dehydrated for 15 min each in a series of ethanols: 50%, 70%, 80%,
90%, 95% and 100% (twice), and then finally 100% overnight at room
temperature. Samples were then rinsed in ethanol followed by rinsing in
propylene oxide and embedded in scipoxy 812 resin with dodenyl succinate
anhydride and nadric methyl anhydride. Semithin sections (0.5 µm) were
obtained and then thin sections (50 nm) were obtained and grid stained with 4%
uranyl acetate and 2.66% lead acetate and observed on an electron
microscope.
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RESULTS |
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To determine when and where Gfi1 is expressed, we carried out in situ hybridization and immunohistochemistry in embryos. As shown in Fig. 1A, Gfi1 mRNA is expressed in E12.5 embryos in the PNS, CNS and many sensory tissues. More specifically, Gfi1 mRNA is expressed in the developing brain, optic epithelia, dorsal root ganglia, otic vesicle, trigeminal and vestibulo-cochlear ganglia, and gut epithelia (Fig. 1A). At later stages expression is also seen in many sensory organs such as the developing eye (Fig. 1B), presumptive Merkel cells (Fig. 1D,E), cells of the nasal epithelia (Fig. 1G), epithelia of the tongue (Fig. 1H), as well as in small clusters of neuroepithelial precursor cells in developing lung (Fig. 1I; D. W., unpublished), and many cells of the developing thymus (Fig. 1K). Hence, Gfi1 mRNA is widely expressed in epithelia in which sensory cells are specified (tongue, nasal epithelia, gut, lung and eye), as well as in the developing brain and PNS ganglia. However, the Gfi1 protein has a more restricted expression pattern and localizes to several specialized sensory cells of the PNS. Gfi1 protein is present in the eye (Fig. 1C), the presumptive Merkel cells (Fig. 1F) and the lung (Fig. 1J). We did not detect Gfi1 protein expression in the brain or any of the ganglia, which is where we see Gfi1 mRNA expression (see Discussion).
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Gfi1 mutant mice display behavioral defects
We previously established a mouse line deficient for Gfi1 (H.
Hock, M. J. Hamblen, H. M. Rooke, D. Traver, R. T. Bronson, S. Cameron and S.
H. Orkin, unpublished). This Gfi1 allele has part of the 5' UTR
along with the entire first and second exon, as well as part of the third exon
deleted. This deleted region contains the entire SNAG transcriptional
repression domain but not the zinc fingers and creates a severe
loss-of-function or null allele. We have found heterozygous mice to be
phenotypically indistinguishable from wild-type littermates at all stages in
our assays. The mutant mice are viable for 3-6 months. The mutants look
similar to wild type and heterozygous littermates until about postnatal day 10
(P10). By P10, the mutants no longer continue to grow at the rate of their
littermates (H. Hock, M. J. Hamblen, H. M. Rooke, D. Traver, R. T. Bronson, S.
Cameron and S. H. Orkin, unpublished;
Karsunky et al., 2002b) and
ataxic behavior becomes apparent. This ataxia and the differences in size
between mutant and heterozygous littermates increase in severity with age (see
video at
http://flypush.imgen.bcm.tmc.edu/lab/deeann/mouse-video1.avi).
The mutant animals display several behavioral abnormalities suggestive of
inner ear defects including hyperactive circling, head tilting, ataxia and
lack of a proper startle response to loud noises. This phenotype coupled with
Gfi1 expression in the inner ear sensory epithelia suggests inner ear
defects.
Gfi1 is required for proper differentiation of hair
cells
As the overall gross morphology of the inner ear appeared normal at P0 in
Gfi1 mutant mice (data not shown), we immunocytochemically stained
ear epithelia with several hair cell markers to identify differentiation
defects. Myosin VI/VIIa is an early marker for hair cell differentiation and
initiation of expression of this marker occurs properly at E13.5 in the mutant
balance organs and cochlea (Fig.
3A,B) (Hasson et al.,
1997). However, as shown in
Fig. 3A,B, the mutant hair
cells in the utricle are thinner and more elongated than the wild-type cells,
and there are two to three layers of myosin VI/VIIa-positive cells in the
mutant instead of the single layer observed in wild-type epithelia.
Anti-myosin VI/VIIa staining of the utricle at E14.5
(Fig. 3C,D) also shows abnormal
hair cell morphology and layering in the mutant. The vestibular organs
normally show a straight line of hair cells at the edge of the lumen, but the
mutant hair cells do not form this straight line as the hair cells are present
in the support cell layer. Similarly, the organization of the auditory hair
cells in the organ of Corti is also aberrant. The characteristic three rows of
outer hair cells and single row of inner hair cells are not present in the
mutant. Serial sections of the organ of Corti often show that one of the outer
rows of cells is lacking as shown with anti-myosin VI/VIIa staining at E16.5
(Fig. 3E,F). Similarly,
Math1 in situ hybridization at E17.5
(Fig. 3G,H), and Brn3c
in situ hybridization at E18.5 (Fig.
3I,J) each show one inner hair cell and two outer hair cells.
Thus, though some hair cells appear to be missing, hair cell-specific markers
are expressed and maintained throughout inner ear hair cell development in
Gfi1 mutant mice. In addition, we find that the remaining mutant hair
cells in the organ of Corti are not properly innervated. As shown in
Fig. 3K,L, staining with
anti-TUJ1, a marker for ß-tubulin in neurons, reveals a very different
staining pattern in mutant animals. In wild-type hair cells
(Fig. 3K), the cochlear neurons
synapse with the base of the three outer hair cells forming a cup-like
staining pattern at the base of each outer hair cell (indicated by green
arrows). In the mutant outer hair cells, the anti-TUJ1 labels the entire cell
body including the cytoplasm. However, the base of the cells where synaptic
sites are normally seen as cup-like structures in wild-type embryos are not,
or are barely visible (indicated by green arrows in
Fig. 3L). This aberrant pattern
is not observed for the inner hair cells. Hence, as the cytoplasm of the outer
hair cells in Gfi1 mutants stain with TUJ1 antibody, the outer hair
cells express a neuronal marker that is not normally expressed in these cells.
We conclude that based on aberrant morphology of vestibular hair cells and
ectopic expression of the neuronal marker TUJ1 in outer hair cells, hair cell
differentiation is affected in Gfi1 mutants.
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Is Gfi1 expression dependent on Math1 expression
and vice-versa?
In flies, proneural gene expression is required for initiation of
senseless expression, and senseless expression is required
for maintenance of proneural gene expression
(Nolo et al., 2000;
Frankfort et al., 2001
). To
determine if a similar relationship exists between Math1 and
Gfi1, we investigated the expression of Gfi1 in
Math1 mutants and Math1 in Gfi1 mutants. As
Math1 and Gfi1 expression overlap temporally and spatially
in the inner ear epithelia, Math1 may be required for Gfi1
expression. We therefore tried to assess Gfi1 expression at the
earliest stage of development, just prior to hair cell formation when hair
cell precursors are specified (E12.5). At this stage, Gfi1 mRNA
expression in the Math1 mutant is present in the entire otic
epithelia similar to wild-type controls
(Fig. 4A,B). However, Gfi1
protein expression is drastically reduced or absent
(Fig. 4C,D). These observations
indicate that initiation of Gfi1 mRNA expression is not dependent on
Math1, but that Gfi1 protein expression is Math1 dependant.
At later stages in development (E14.5, E16.5 and E18.5), Gfi1 mRNA
and protein are both drastically reduced or absent in Math1 mutants
(Fig. 4E,F and data not shown).
This may imply that Math1 is required for Gfi1 expression,
or, alternatively, that the cells in which Gfi1 is expressed are not
specified (Chen et al., 2002
).
It also indicates that Gfi1 mRNA and protein expression is confined
to hair cells at later developmental stages.
|
Is Gfi1 required to maintain Math1 expression? To monitor
Math1 expression in Gfi1 mice, we used a mouse containing
the ß-galactosidase-coding region that replaced the entire
Math1-coding region. ß-galactosidase staining of heterozygous
Math1 animals faithfully mimics Math1 expression, whereas
hair cell specification appears normal
(Ben-Arie et al., 2000). Mice
that were Math1 heterozygous
(Math1+/ßGal) and Gfi1 wild-type or
null mutant were derived. In the Gfi1 mutants,
Math1ßGal expression is present in all inner
ear sensory epithelia (Fig.
4G-J, Fig. 5A-L).
However, sections of P0 saccules stained with ß-galactosidase show some
disorganization and some cells appear to be present in the supporting cell
layer (Fig. 4G,H). The mutant
hair cells also seem to have less well organized stereocilli than do wild-type
cells. Note, however, that the cristae appear to be less affected or
unaffected as they have a very similar morphological appearance to wild-type
cristae (Fig. 4I,J). Similar
data were also observed with Math1 in situ hybridization in
Gfi1 mutant organ of Corti (Fig.
3G,H). In summary, we found no obvious changes in Math1
expression pattern in Gfi1 mutants. These data suggest that there is
no dependence of Math1 on Gfi1 in mouse. This is in contrast
to what we observed in fruit fly between atonal and
senseless (Frankfort et al.,
2001
; Nolo et al.,
2000
).
|
Gfi1 is required for cochlear hair cell survival
The Math1ßGal/+; Gfi1 mice provided us
with a convenient tool to follow hair cell development in wholemounts of organ
of Corti and asses differences in apical and basal areas of the cochlea.
Normally, by E15.5 Math1/ß-galactosidase expression is visible
in the developing hair cells of the organ of Corti and rows of hair cells are
beginning to differentiate in a basal-to-apical gradient. As shown in
Fig. 5A,B, by E15.5,
Math1/ß-galactosidase positive cells are present in wild-type
and mutant embryos. However, the rows are not as clearly defined in the mutant
as in the wild type. By E17.5, the wild-type hair cells have formed the
characteristic one row of inner hair cells and three parallel rows of outer
hair cells (Fig. 5C). At E17.5,
the mutant hair cells are disorganized and less numerous in the basal cochlea
(Fig. 5D). As shown in
Fig. 5E-H, by P0 the loss of
hair cells has progressed in a basal to apical gradient.
Fig. 5E shows the orderly
arrangement of wild-type hair cells at P0, whereas the basal cochlea of the
Gfi1-null mice has lost the majority of its hair cells as gauged by
Math1 expression (Fig.
5F). The medial cochlea is also severely affected, but the inner
hair cells are still present (Fig.
5G). At P0 the apical cochlea shows little to no loss of hair
cells, but does exhibit a disorganization similar to that seen in basal
cochlea as early as E15.5 (Fig.
5H, compare with Fig.
5B). By P3 the loss of hair cells in the basal to apical gradient
is more severe in the mutants (Fig.
5I-L). The basal cochlea has few hair cells by P3
(Fig. 5J). The medial cochlea
still retains the majority of inner hair cells but has lost almost all outer
hair cells (Fig. 5K). Even the
apical cochlea is beginning to show drastic reduction in the number of hair
cells by P3, though again it appears as though the outer hair cells degenerate
first. Hence, it appears that the inner and outer hair cells are initially
specified by E15.5 but are subsequently lost in a basal-to-apical gradient. In
all cases, the outer hair cells in a given region degenerate prior to the
inner hair cells.
As the mutants age, the organ of Corti becomes unrecognizable. Analysis of serial sections stained with Hematoxylin and Eosin indicates that by P14 all cochlear hair cells and most support cells have disappeared in mutant animals (Fig. 6A,B). However, despite the rapid degeneration of cochlear hair cells, the hair cells in the vestibular organs do not degenerate, but remain unorganized (Fig. 6C,D). Note the separation of hair cells and support cells in the saccule of the wild-type mouse (Fig. 6C). This layering is again not as clearly defined in the mutant when compared with wild type (Fig. 6D).
|
As shown in Fig. 2D
(asterisk), Gfi1 mRNA is also expressed in the cochlear ganglion
neurons, although we did not observe Gfi1 protein expression. We therefore
examined number and morphology of the cochlear ganglion neurons. As shown in
Fig. 6E,F, at P7, both
wild-type (Fig. 6E) and mutant
(Fig. 6F) cochlear ganglion
neurons show similar cell densities and a low level of apoptosis as indicated
by anti-activated-caspase 3 (C3) staining. Low levels of apoptosis at P7 in
wild-type cochlear ganglion neurons has been previously observed
(Kamiya et al., 2001). At P21,
the cochlear ganglion neurons in the wild-type
(Fig. 6G) and mutant mice
(Fig. 6H) show slightly
different cell densities and quite a few mutant cells express
activated-caspase 3, suggesting that these cells undergo cell death by
apoptosis. We did not observe apoptosis in the wild-type mouse at P21. By 5
months of age there is a dramatic reduction of neurons in the cochlear
ganglion, as seen by Hematoxylin and Eosin staining
(Fig. 6I,J). Hence, cochlear
ganglion neuron degeneration occurs after hair cell loss and is
progressive.
Ultrastructural analysis of the organ of Corti
To determine the ultrastructural defects in the cells of the organ of
Corti, we carried out transmission electron microscopy (TEM). TEM shows
abnormal hair cell morphology at E18.5 in the mutant mice and confirms the
disorganization of hair cells in the organ of Corti. The stereocilli are
nicely preserved and easily identifiable in the wild type outer hair cells
(Fig. 7A,C), but the
stereocilli of the mutant hair cells are not well preserved and are barely
visible in some cells. Some of the mutant outer hair cells also display
typical morphological signs of apoptosis, such as shrinkage of the cell body,
extensive blebbing and vacuolization (Fig.
7B,D). Also, consistent with an apoptotic mechanism of cell death,
the mutant mitochondria appear indistinguishable from the wild-type
mitochondria, in contrast to what happens when cells die by necrosis. These
data suggest that the outer and inner hair cells die by apoptosis.
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DISCUSSION |
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Gfi1 mRNA is present in a variety of tissues during development.
It is expressed in the CNS, a variety of ganglia and many specialized sensory
cells of the PNS. However, the Gfi1 protein expression pattern is more
restricted. We detect Gfi1 protein primarily in specialized sensory cells of
the PNS. However, we did not detect Gfi1 protein in the CNS or any ganglia.
The difference in Gfi1 mRNA and protein expression may have several
possible explanations. First, senseless mRNA is also more widespread
than its protein expression pattern in the fly
(Nolo et al., 2000). Thus, it
is likely that this is a real phenomenon and not just an artifact of in situ
or immunohistochemical analysis. Alternatively, the RNA and/or the protein
stability may vary from cell to cell type. Third, it is possible that Gfi1 is
only translated under specific conditions, i.e. in the presence of Math1.
The temporal and spatial distribution of Gfi1 transcripts in many
cells and epithelia overlaps with that of many bHLH gene expression patterns.
In most tissues in which Gfi1 is expressed, there is a corresponding
bHLH gene that may regulate/control Gfi1 expression. For example,
Mash1 (Ascl1 Mouse Genome Informatics) is expressed
in the developing olfactory epithelia (Cau
et al., 1997), the neuroendocrine cells of the lung
(Borges et al., 1997
;
Ito et al., 2000
) and the
tongue (Seta et al., 1999
),
where we observe Gfi1 mRNA expression. Math1 is expressed in
the developing ear epithelia (Bermingham et
al., 1999
), gut epithelia
(Yang et al., 2001
) and Merkel
cells (Ben-Arie et al., 2000
)
where we observe Gfi1 mRNA and protein expression. Math5 is
expressed in the developing eye and retinal ganglion cells
(Wang et al., 2001
).
Neurod1 is expressed in the ear epithelia, as well as the ganglia
that innervate the ear (Liu et al.,
2000
). These bHLH genes are homologous to the Drosophila
bHLH proteins Achaete, Scute, Atonal or Amos and are required for the
specification of subtypes of cells. Similarly, other bHLH proteins such as the
neurogenins have been shown to be expressed and required in some of the PNS
ganglia where Gfi1 mRNA is expressed
(Ma et al., 1997
;
Ma et al., 2000b
). These
observations suggest that, similar to fruit flies, Gfi1 expression
may be regulated by bHLH genes.
Because we found Gfi1 to be expressed in the developing ear, we
chose to focus our analysis on the developing ear to test potential
interactions of Gfi1 with the bHLH gene Math1
(Bermingham et al., 1999). We
assessed the expression pattern of Gfi1 in Math1 mutants and
Math1 expression in Gfi1 mutants. Interestingly,
Math1 expression is unaffected in Gfi1 mutants. As atonal
positively regulates its own expression in the fly
(Sun et al., 1998
), it is
possible that Math1 may also regulate its own expression in the
mouse. This provides a potential explanation as to how Math1
expression may be maintained in the Gfi1-deficient mouse. This data
may also suggest that Gfi1 is not required for maintenance of
Math1 expression. However, Math1 is required for Gfi1
protein expression, but not required for initial Gfi1 mRNA
expression. Hence, it remains to be established how Gfi1 mRNA and
protein expression is precisely controlled.
Our data support a model where Gfi1 is downstream of Math1, but
may not support a model in which Gfi1 functions in a positive
feedback loop with Math1. However, it is possible that another bHLH
gene expressed in the vertebrate ear epithelium prior to Math1
expression is required for Gfi1 mRNA expression. The identity of this
putative bHLH protein is unknown. However, the existence of such factor is
suggested because in Math1-null mutants, hair cell precursors form a
zone of non-proliferating cells that delineate the sensory primordium within
the cochlear anlage, and a significant subpopulation of these precursors die
because of apoptosis in a basal-to-apical gradient
(Chen et al., 2002). The fact
that these cells die instead of becoming support cells indicates that these
cells have a different fate than their surrounding cells in the absence of or
prior to Math1 expression. We surmise that this difference is induced
by the presumptive factor. Our data are consistent with the idea that this
factor is upstream of both Math1 and Gfi1. Such a factor
could function similar to a proneural gene as it might be initially expressed
in a cluster of cells rendering them competent to become neural cells and then
refine to a specific cell that also expresses Math1 and Gfi1
to become a hair cell (Chen et al.,
2002
; Hassan and Bellen,
2000
). This factor could be responsible for the initial expression
of Gfi1 and explain why in a Math1 mutant we observe
Gfi1 mRNA expression early on. Candidate bHLH transcription factors
expressed prior to Math1 in ear development include Neurod1
(Liu et al., 2000
) and
neurogenin 1 (Ma et al.,
2000a
). Both are required for proper development of the inner ear,
but Neurod1 and neurogenin 1 mutant mice display very different
phenotypes from the ones we observe in Gfi1 mutants
(Liu et al., 2000
;
Ma et al., 1998
), suggesting
that neither Neurod1 nor neurogenin 1 corresponds to the proposed
factor.
Gfi1 is required for hair cell development in the vestibule
and hair cell differentiation and viability in the organ of Corti
The hair cells of the inner ear seem to be specified properly as they
express many of the typical hair cell markers such as myosin VI/VIIa,
Math1 and Brn3c. Thus, Gfi1 is not required for the
specification of hair cells as they are formed in both the vestibule and the
cochlea. However, the loss of Gfi1 seems to affect the vestibular and
cochlear hair cells differently. In the vestibule, the hair cells are
morphologically abnormal at the earliest stages of hair cell differentiation
and at all subsequent stages. In addition, hair cells are not specifically
localized to a lumenal monolayer, and are more variable in size and shape.
This disorganization of hair cells in the vestibule may account for the ataxic
behavior of the mice. In the cochlea, Gfi1 is required for the
organization and maintenance of both inner and outer hair cells. Although the
mutant hair cells seem to be specified in the developing organ of Corti as
early as E15.5 and express typical hair cell markers, they are disorganized.
In addition, the outer hair cells express the neuronal marker TUJ1 at E17.5.
This abnormal/ectopic TUJ1 expression may indicate a partial transformation of
outer hair cells into neurons, or the de-repression of a single neuronal
marker. It is thus possible that these cells are part hair cell and part
neuron, and this ambiguity could trigger apoptosis. In fact, the outer
cochlear hair cells are the first to disappear starting at E17.5. Based on TEM
analysis, we see some of the classical morphological signs of apoptosis in the
mutant hair cells at E18.5, including shrinkage of the cell body, blebbing and
vacuolization. Whole-mount analysis of the cochlea indicates that this loss of
hair cells occurs in a basal to apical gradient and affects outer hair cells
prior to inner hair cells in any given region of the cochlea. The hair cells
and support cells of the organ of Corti continue to disappear until the entire
organ of Corti has been destroyed by P14. Because wild type mice do not
perceive sound until after P12 (Kamiya et
al., 2001), and because Gfi1 null mice have no hair cells
by P14, we assume that these mice are deaf, which is in agreement with the
lack of a startle response to loud noises.
Upon degeneration of the organ of Corti, the cochlear ganglion neurons also
degenerate. This degeneration is progressive, beginning after P7 but fairly
extensive by five months of age. As Gfi1 mRNA is expressed at low
levels in the neurons, it may be directly required for neuronal survival.
However, Gfi1 may not be directly responsible for neuronal loss
because Gfi1 protein is not expressed in the cochlear ganglion, and cochlear
neurons normally die after degeneration of hair cells, presumably because of
the withdrawal of trophic support (Dodson,
1997; Lefebvre et al.,
1992
). Thus, it seems likely that the loss of cochlear ganglion
neurons is secondary to the loss of hair cells in the organ of Corti.
There are other mutant mice with similar, yet distinct phenotypes.
Brn3c-deficient mice have a similar vestibular phenotype with a small
number of hair cells retained in the support cell layer in the vestibular
sensory epithelia (Xiang et al.,
1998). The hair cells in Brn3c-null mice are also
initially specified, but fail to mature and form stereocilli. The
Brn3c-deficient hair cells then rapidly degenerate by apoptosis
(Xiang et al., 1997
). The loss
of hair cells occurs in the organ of Corti as early as E17.5 with nearly
complete loss by P5. This is similar to the Gfi1 mutant, but unlike
in the Gfi1 mutants, this degeneration is also detected in the
vestibule as early as E18.5. In Brn3c mutants, the loss of hair cells
is then followed by a loss of the cochleo-vestibular neurons with a
substantial loss as early as P4, earlier than in the Gfi1 mutants.
Note that Brn3c expression is maintained in all inner ear sensory
epithelia of Gfi1 mutant mice
(Fig. 3I,J; data not shown).
Barhl1-deficient mice also show a progressive degeneration of
cochlear hair cells (Li et al.,
2002
). This degeneration is much slower than in Gfi1
mutants, occurring roughly from P6 to 2 months of age for outer hair cells and
between six months and ten months for inner hair cells. Interestingly, the
outer hair cells degenerate first in an apical-to-basal gradient, and the
inner hair cells degenerate second in a reverse basal-to-apical gradient.
Gfi1 has a novel phenotype with respect to its effect on the inner
ear. The fact that different types of hair cells expressing Gfi1 in
the different sensory organs are affected differently is mirrored in
Drosophila senseless mutants and C. elegans pag-2 mutants.
In the Drosophila embryonic PNS, some mutant neuronal subtypes
undergo apoptosis like the auditory hair cells in Gfi1 mutants
(Nolo et al., 2000). In C.
elegans, other neuronal subtypes are improperly differentiated or
abnormal such as the BDU interneurons, the Pn.aa neuroblasts, and the VA and
VB motoneurons similar to the vestibular hair cells in the Gfi1
mutants (Cameron et al.,
2002
).
Hence, it is possible that the function of Gfi1 and its homologs is dependent on the tissue in which it is expressed as it may have a variety of functions depending on its environment. It is most likely that Gfi1 plays a variety of different roles as a transcriptional repressor or activator. Thus, different genes are repressed or activated in different tissues resulting in a variety of functions. A more precise explanation as to the function of Gfi1 will have to await the further analysis of Gfi1 function in other tissues and the identification of direct Gfi1 target genes and interaction partners.
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ACKNOWLEDGMENTS |
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