Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA
* Author for correspondence (e-mail: dfekete{at}purdue.edu)
Accepted 1 February 2005
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SUMMARY |
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Key words: Labyrinth, Placode, Otocyst, Cell lineage, Neuroblast, Chick
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Introduction |
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It has been proposed that a delaminating neuroblast may carry positional
information to direct its neurite back to the sensory region from which it
delaminated (Fritzsch et al.,
2002). This suggests that neurons might recognize their synaptic
partners because they are clonally related. Although dye labeling of the
zebrafish lateral line failed to confirm this hypothesis
(Gompel et al., 2001
),
definitive studies for other mechanosensory organs are lacking. We sought to
address this issue in two classes of mechanosensory organs located in the
middle and inner ears of the bird. The inner ear poses a particular challenge
for neuronal targeting, as, here, a single placode gives rise to two general
classes of neurons (vestibular and auditory) and multiple different sensory
organs. In the bird ear, this includes four maculae, three cristae and one
auditory organ (the basilar papilla). The logistics of how neurons sort to
their correct targets in the periphery could, in theory, be simplified if the
neuroblasts leave the otic ectoderm with information that allows them to
project back to the progeny of their own sister cells. A lineage relationship
between auditory neurons and the basilar papilla could not be confirmed in a
prior study where progenitors were targeted at otocyst stages in chick
(Lang and Fekete, 2001
). This
study seeks evidence that bipotent neurosensory progenitors might be present
at earlier stages of otic development.
The molecular basis of cell-fate specification between the two classes of
inner ear neurons, auditory and vestibular, is unresolved. Expression of the
transcription factor, Gata3, distinguishes neuroblasts apparently destined for
auditory (Gata-3+) versus vestibular (Gata3) ganglion fates even before
they emigrate from the otocyst in mouse
(Lawoko-Kerali et al., 2004).
This early segregation could provide a mechanism by which the neuroblasts
acquire different identities before delaminating
(Fekete and Wu, 2002
).
Inner ear lineages are also of interest in the context of evolutionary
homologies. For inner ear mechanosensory organs, the issue has been raised of
whether they share an ancient evolutionary relationship to insect
mechanosensory organs (Eddison et al.,
2000; Fritzsch et al.,
2000
; Lewis,
1991
). Both systems use Notch-Delta signaling during key stages of
neuroblast delamination (Eddison et al.,
2000
) and Atonal homologs for cell fate specification
(Bermingham et al., 1999
;
Chen et al., 2002
;
Zheng and Gao, 2000
). In the
invertebrate organs, mechanosensory cells and their supporting cells share a
common lineage with the neurons that supply the innervation
(Gho et al., 1999
;
Hartenstein and Posakony,
1989
). It could be argued, then, that finding a shared lineage
between neurons and sensory cells in vertebrate mechanosensory organs further
strengthens the developmental parallels between vertebrate and invertebrate
mechanosensory organs.
Gene expression patterns indicate extensive regionalization of the ear by
the otocyst stages in several vertebrate classes (reviewed by
Fekete, 1999;
Riley and Phillips, 2003
;
Torres and Giraldez, 1998
).
This pre-patterning may serve to specify the major parts of the ear,
segregating them into separate lineages. By contrast, fate mapping in
Xenopus showed that cells originating from restricted regions of the
otic vesicle can colonize widely separated sensory organs
(Kil and Collazo, 2001
). It is
therefore essential to conduct clonal analysis on a higher vertebrate, to ask
whether the cell dispersion seen in the frog is a general feature of
developing inner ears.
This lineage study was initiated to address three questions. Does each neuron originate from the same focal part of the sensory primordium that it will subsequently innervate? Is there a separate origin of auditory versus vestibular neuroblasts? And can clones disperse across the different subdivisions of the inner ear? We find that in the middle ear, a clonal relationship between the ganglion and the sensory organ was common. In the inner ear, a shared lineage between neurons and sensory organs of the inner ear is also possible, although it is less common than separate lineages. Furthermore, an individual neuron is not necessarily related to the sensory organ it innervates in the ear. The auditory and vestibular neurons can originate from a shared lineage, although the majority derive from separate lineages. Finally, we find that individual clones do not colonize more than one anatomical subdivision of the inner ear, with the exception of clones that include ganglion neurons.
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Materials and methods |
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Injection and detection of viruses
All embryos were assigned stages (s) according to Hamburger and Hamilton
(Hamburger and Hamilton,
1951). Virus was delivered to the otic cup (stages 9.5-15) or otic
vesicle (stages 16-18) of SPAFAS standard White Leghorn chicken embryos as
described (Homburger and Fekete,
1996
; Lang and Fekete,
2001
). Embryos were fixed at E9-13 in 4% paraformaldehyde in PBS
at 4°C for 30-60 minutes, washed with PBS and frozen-sectioned at 25-35
µm. Slides were processed for ß-galactosidase or alkaline phosphatase
histochemistry (Fekete et al.,
1994
). Selected sections were stained with Hoechst 33342 and 1:500
mouse anti-NF160 (Sigma) followed by anti-mouse
IgG1-Alexa-fluor-488 (Molecular Probes).
After removing coverslips, AP+ cells and a minimal amount of surrounding
tissue were collected with 30 gauge needles under a dissection microscope.
Similar sized fragments were picked from AP-negative regions as controls. PCR
amplification and sequencing of the variable region of CHAPOL proviral DNAs
were carried out as described (Golden et
al., 1995).
Electroporation of plasmid DNA into chicken otic cup
Episomal marker plasmid, pRep4-CMV-GVP-H2B-EYFP, was constructed from pREP4
(Invitrogen) as the backbone by inserting DNA fragments of cytomegalovirus
immediately early promoter from pDsRed1N1 (Clontech), a fragment containing
Gal4-VP16 gene, 14 repeats of UAS and E1b promoter from EFGVPUlynUH2B
(Koster and Fraser, 2001) and
human Histone2B (Kanda et al.,
1998
) fused with EYFP (Clontech). Plasmid
pRep4-CMV-GVP-H2B-EYFP was introduced to the right otic cup of 26 embryos
(stages 11-14) using electroporation. An epoxy-insulated tungsten
microelectrode with 20 µm exposed tip was placed on the right otic
epithelium as the cathode, and a 0.5 mm tungsten wire anode was placed to the
left of the head. The otic cup was filled with 1 µg/µl plasmid using a
beveled glass micropipette attached to the Picospritzer. Electroporation
consisted of five square pulses of 10-20V, 1 mseconds each, applied using a
BTX T820 pulse generator. On E9, heads from 15 surviving embryos were fixed
with 4% paraformaldehyde in PBS for 45 minutes on ice, frozen, sectioned at 50
µm and stained with 1:100 Alexa-fluor-564-phalloidin (Molecular Probes) and
1:500 mouse anti-NF160 (Sigma) followed by anti-mouse
IgG1-Alexa-647 and TOTO3 (Molecular Probe). EYFP-positive nuclei in
the statoacoustic ganglia were counted using a Nikon E800 microscope with 10
x objectives, and selected nuclei were analyzed three-dimensionally to
judge anatomical identity using MRC1024 confocal microscope with 60 x
objective in z-series optical sections.
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Results |
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We generated a pseudotyped virus stock carrying a VSV-G envelope around the Rous Sarcoma Virus (RSV)-based protein core. Packaged within this were two different replication-defective viral genomes, CHAPOL and RDlac encoding human placental alkaline phosphatase (AP) and ß-galactosidase, respectively. We compared results using this pseudotyped mixed virus stock to a stock carrying virions of similar genotypes coated instead with the RSV A-subgroup envelope protein. However, the two virus stocks differed nearly sevenfold in concentration, with the A-subgroup stock having the higher titer (6.7 x106 versus 1 x106 infectious units/ml).
Viruses were delivered to the otic cup or vesicle in 147 embryos. For each virus subgroup, we examined 3-41 embryos per stage (mean=15 embryos per stage). Ears were scored as positive if they showed histochemical staining within the otic epithelium or its ganglia for either AP or ß-galactosidase on embryonic days 7-10 (E7-E10). Injections with G-subgroup viruses at stages 14, 15, 16, 17 and 18 yield positive ears in 17%, 57%, 40%, 85% and 100% of cases, respectively. By comparison, A-envelope viruses of higher titer delivered at the same stages yield clones in 0%, 0%, 11%, 33% and 70% of injected ears. These results indicate that VSV-G pseudotyped viruses are considerably more effective as reagents for lineage analysis in the chick inner ear.
Distribution of AP+ cells
For lineage analysis, we targeted the ear as early as possible to try to
generate large and potentially more varied clones. We used CHAPOL(G) virus at
titers of 1 x106 i.u./ml (131 embryos) or 4.5
x106 i.u./ml (147 embryos). Virus was delivered near the otic
cup at stages 9.5-14.5, and the ears were processed on E10-13. Out of 278
injected ears, 80% have at least one AP-positive (AP+) cell in the
paratympanic organ, inner ear epithelium or the associated ganglia.
AP+ cells are unevenly distributed throughout the various subdivisions of the inner ear, as summarized in Fig. 1. They are found most frequently in the non-sensory part of utricle and the endolymphatic apparatus, including both the duct and the sac. We separately scored AP+ cells located in sensory versus non-sensory regions of the major anatomical subdivisions of the ear. In total, 18% of injected embryos have sensory clones. The frequency of infecting any single sensory organ varies as follows: paratympanic organ>basilar papilla>utricular macula>superior crista>saccular macula>lagenar macula> posterior crista>lateral crista. There is no obvious variation in the sensory organ labeling with respect to the stage of injection, with the possible exception of the lagenar macula, which is labeled only by early injections (stage 10.5-11). The statoacoustic ganglia are labeled with moderate efficiency at all injection stages, as are non-sensory cells in the remaining parts of the labyrinth. Owing to the method of injection, infected cells are not restricted to the inner ear, but can also include other ectodermal derivatives. For example, AP+ cells are found in the epidermis, the trigeminal ganglion, the geniculate ganglia and the paratympanic organ.
|
Clones are usually restricted to distinct anatomical parts of the inner ear
The virus stock, CHAPOL(G), is a library of replication-incompetent vectors
that each carry a 24 bp tag in addition to the AP gene. Clonal analysis of
infected ears was performed by amplifying and sequencing the 24 bp variable
fragment independently from one or a small cluster of AP+ cells. We selected
29 ears of greatest interest, primarily because they contained AP+ cells in
both sensory organs and ganglia. From these, 629 small pieces of tissue
(picks) containing one or more AP+ cells were removed for analysis. Slightly
less than half (306 picks) were successfully amplified by PCR and sequenced.
By comparison, PCR product was amplified from only three out of 52 AP-negative
picks chosen based on proximity to AP+ tissue. Among the sequenced PCR
products, 40 contain more than one library sequence and were not considered
further. Multiple sequences are thought to originate from progenitor cells
that accept more than one virion. In total, sequencing of AP+ picks yielded
139 unique sequences or clones. None of the picks has an identical sequence
with picks from other ears, suggesting that viruses carrying any single tag
are not over-represented within the library.
The distribution of successfully amplified picks and the resulting clonal relationships are shown schematically in Fig. 1B, with specific examples in Fig. 1C-F. Of the confirmed clones, the vast majority (124/139) do not extend beyond a single anatomically defined structure in the inner or middle ears. The 15 clones identified as having members colonizing more than one structure will be discussed in detail later. In summary, three out of the 15 are associated with the middle ear and geniculate ganglion. Five clones show members both in the inner ear and one or both of its ganglia and seven clones are dispersed across the two otic ganglia. Among clones restricted to the inner ear epithelium, only a single clone contains cells located in two anatomically distinct structures. In this case, clonally related cells are present in the superior ampulla and the adjacent part of the superior semicircular canal (Fig. 1C). In all other ears, AP+ cells in different anatomical parts of the inner ear are clonally independent, even if they are located in close proximity to each other.
Despite the early injection paradigm, only 14% (39 of 278) of ears have AP+ cells located in inner ear sensory organs. Only a fraction of those, a scant four ears, has AP+ cells in multiple sensory organs. Successful sequencing of three of these cases failed to indicate a clonal relationship between different sensory organs (basilar papilla and utricular macula, n=2; basilar papilla, utricular macula, superior crista and lateral crista, n=1; see Fig. 1B). The probability of finding more than two independent viral infections in sensory epithelia can be estimated using Poisson regression analysis with the assumption of independence of each sensory organ (not shown). The estimated frequency of 1.35% matches the observed frequency of 1.40%. The distribution of inner ear sensory clones supports the hypothesis that progenitors colonizing each sensory organ may already be separated by the time the proviral DNA integrates into the host cell genome.
Neurons and sensory organs can share a common progenitor in the middle ear
Sixteen ears have AP+ cells in the paratympanic organ. Nine out of these
(56%) also have AP+ cells in the geniculate ganglion that resides immediately
adjacent to the organ and is the source of its innervation
(Fig. 1). We successfully
tested the clonal relationships in five of them, and found three cases where
AP+ cells in the paratympanic organ and geniculate ganglion are clonally
related (Fig. 2). If we
extrapolate the sequencing results to include the unsuccessful cases, we
estimate that approximately one-third (5.4/16) of ears with infection of the
paratympanic organ will show evidence of a shared neurosensory lineage. No
clonal relationship was found between the geniculate ganglion and either the
inner ear or the statoacoustic ganglion in nine tested cases. In one case
(Fig. 1F), neuronal cells in
the facial nerve (between the geniculate and vestibular ganglia) are clonally
related to the cells in geniculate ganglion. We have no samples where AP+
cells spanned both the sensory and non-sensory epithelium of the paratympanic
organ (not shown). We conclude that on or shortly after E2, there are
multipotent epibranchial placode progenitors that give rise to both sensory
cells and neurons.
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Neurons and non-sensory epithelium can share a common progenitor in the inner ear
Although some otic neurons can be related to the utricular macula sensory
organ, we also found that they can share a lineage with non-sensory cells
adjacent to the utricular macula. In two out of three tested ears, cells in
the acoustic ganglion are clonally related to non-sensory epithelial cells
residing between the utricular macula and the lateral crista
(Fig. 4). We refer to this
region as the utricle-lateral ampulla junction (ULJ). Its identity as
non-sensory is indicated by the absence of neurofilament-positive nerve fibers
penetrating into the epithelium (not shown). Except for this region, we found
no other clonal relationships between neurons and non-sensory epithelium
(n=10, Figs 1 and
2). Furthermore, in no case are
non-sensory regions related by lineage to sensory organs.
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On E9, electroporated ears have EYFP+ cells that are widely distributed in the ear epithelium, ganglia and nearby epidermis on the right side. The Gal4-UAS cassette made it considerably easier to distinguish EYFP+ cells from negative cells in comparison with cells labeled with plasmids lacking the Gal4-UAS cassette (data not shown). No EYFP+ cells are found in the periotic mesenchyme, confirming that plasmid does not penetrate beyond the surface ectoderm under these electroporation conditions.
Nine out of 15 specimens contain EYFP+ cells in the statoacoustic ganglia. These ears were processed for fluorescent labels to distinguish neurons from satellite cells. The cytoplasm of satellite cells (and Schwann cells) is rich in F-actin and can be readily stained by phalloidin conjugated to a fluorochrome. By contrast, neuronal somas have a paucity of F-actin but instead are stained by anti-neurofilament-160 (Fig. 5C). We used confocal microscopy to observe 128 EYFP+ cells in these ganglia. The vast majority (n=122) clearly have a neurofilament-enriched cytosol surrounding their EYFP+ nucleus (an example in Fig. 5D), while none of the remainder are heavily stained by phalloidin. These data provide independent confirmation that only the neurons, and not their associated glial cells, arise from the otic placode. Therefore, from our retrovirus lineage study, we can conclude that the AP+ cells in the otic ganglia found to be clonally related to other otic placode derivatives, such as the inner ear epithelial cells, are indeed neurons and not glial cells.
Acoustic and vestibular ganglion neurons can share a common progenitor
In 28 ears with AP+ cells in the acoustic ganglion, 22 ears (79%) have AP+
cells in the vestibular ganglion. This strong correlation indicates the
possibility that a shared progenitor pool populates these two ganglia. AP+
cells in both ganglia were picked from 15 ears and analyzed for clonal
relationships. In seven ears, we could confirm that single clones were
dispersed across the acoustic and vestibular ganglia. Three of these are shown
schematically in Fig. 5E-G (red
triangles in each panel). Another is the ear shown in
Fig. 3A that also has utricular
macula members.
There were 22 ears with AP+ cells in both ganglia. If we extrapolate from
our successful sequencing data, 10 of these will have clones that
disperse across both ganglia. The other 12 should have clones that are
restricted to each ganglion (e.g. Fig.
1D). Six ears have clones in the acoustic but not vestibular
ganglion and 36 ears have clones in the vestibular but not acoustic ganglion.
In total, among the ears displaying neuronal clones, we estimate that 84%
(54/64) will have clones that remain confined to individual ganglia.
The avian acoustic ganglion is unusual in that it includes vestibular
neurons projecting to the lagenar macula
(Fischer et al., 1994;
Kaiser and Manley, 1996
). We
therefore had to consider the possibility that some of the AP+ neurons located
in the acoustic ganglia might be lagenar-projecting vestibular neurons.
Electron microscopic analysis (Fischer et
al., 1994
) and tract tracing of lagenar afferents back to their
cell bodies of origin (A. Campero, T.S. and D.M.F., unpublished) both indicate
that lagenar and acoustic ganglion neurons do not spatially overlap. Rather,
lagenar-projecting neurons are located just beyond the medial edge of the
acoustic ganglion, within the large nerve bundle traveling between the lagenar
macula and the vestibular ganglion. Auditory neurons are distributed
throughout the entire acoustic ganglion. We conclude that most of the AP+
cells in the acoustic ganglia that share a lineage with vestibular cells are,
in fact, auditory neurons, based on their spatial distribution within the
ganglion. This indicates that vestibular neurons and auditory neurons can be
clonally related, and that auditory neurons can be related to cells in a
vestibular sensory organ.
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Discussion |
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Distribution of neurogenic and prosensory domains
One of the outstanding lineage questions for inner ear development has been
whether neurons and sensory organs share a common progenitor. A shared lineage
was proposed based on homology to mechanosensory organs in flies, where
neurons, mechanoreceptors and their support cells all arise from a single
sensory organ progenitor (SOP) cell (Adam
et al., 1998). Early during ear development, both lineages express
some of the same neuroblast-associated proteins, including the LIM-homeodomain
protein, Islet1, in the mouse
(Radde-Gallwitz et al., 2004
)
and the homoebox transcription factor, Prox1, in the chicken
(Stone et al., 2003
). Further
circumstantial evidence of a shared lineage can be deduced from gene
expression studies showing spatial and temporal overlap between neurogenic and
prosensory markers (Fig. 6B).
The neurogenic genes, neurogenin1 and NeuroD, identify a
broad neurogenic domain in the ventral otocyst, although they are excluded
from the most posterolateral region, which is occupied instead by cells
expressing an inhibitor of neural fate determination, Tbx1, in the
E9.5 mouse (Raft et al.,
2004
). By E10, neurogenic gene expression is largely co-extensive
with expression of Lunatic fringe (Lfng), a gene that may
identify a sensory-competent zone (Cole et
al., 2000
). Only in the most posterior part of the vesicle does
Lfng expression extend beyond the neurogenic domain
(Fekete and Wu, 2002
;
Raft et al., 2004
). In the
chicken, the combined expression domains of Lfng and
Serrate-1 define a putative sensory-competent region
(Cole et al., 2000
). This
region, and later the prosensory patches, are also marked by the cell-adhesion
molecule BEN (Goodyear et al.,
2001
). Lfng is co-expressed with Fgf10, a gene
that acts upstream of neurogenin1 to regulate the size of the
neurogenic domain in the chicken (Alsina et
al., 2004
). Thus, in both the mouse and the chicken, there is
evidence that neurogenic and the presumed sensory-competent domains are mostly
co-extensive.
How much of the sensory-competent zone actually generates sensory organs
rather than neurons remains unresolved in the absence of a high-resolution
fate map of the region. In general, gene expression data suggest that only a
subset of sensory-competent cells will ultimately generate sensory organs in
the chicken ear. A comparison of Lfng and/or Serrate-1 as
sensory-competent markers with Bmp4
(Cole et al., 2000) and
Prox1 (Stone et al.,
2003
) as prosensory markers suggests that the first sensory
primordia are specified near the anterior and posterior poles of the
sensory-competent zone. The sensory primordia then arise sequentially in
discrete regions of the sensory-competent domain.
It is in the context of these prior studies that we consider our lineage
data, and offer a predicted fate map for the neurogenic, sensory-competent and
prosensory primordia (Fig. 6C).
The last we define as the regions within the sensory-competent zone that will
ultimately form individual sensory organs. Within the sensory-competent
domain, the prosensory regions map mostly along the edges. Neurogenic
progenitors fill in the anterior and central part of the sensory-competent
region, overlapping with prosensory cells only in the region of the primordial
utricular macula, as suggested by our lineage data. This location is
interesting in view of a previous report that ongoing delamination of neurons
from the utricular primordium was observed as late as stage 27 in the chicken
(Stone et al., 2003). There is
also overlap of the neurogenic zone with non-sensory cells in the ULJ; we
presume these cells arise from within the sensory-competent zone, but fail to
acquire a sensory fate.
It is still unclear whether the primordium of the basilar papilla and
lagenar macula (BP/LM) is also neurogenic. Previous studies indicate that most
neuroblasts delaminate from ventral pole of otic vesicle
(Adam et al., 1998;
Alsina et al., 2004
;
Stone et al., 2003
), which is
close to or overlapping with the primordial BP/LM
(Cole et al., 2000
). However,
we failed to find evidence of clonal relationships between acoustic sensory
epithelium and the statoacoustic ganglia in four successfully tested ears. We
can add to these data another five ears derived from a previous lineage study
performed at otic vesicle stages (Lang and
Fekete, 2001
). Because of the small number of samples, we cannot
exclude the possibility of a shared lineage between these two auditory
tissues. It may be that neurogenic progenitors are lineally separated from the
primordial BP at an early stage but remain in the epithelium for several days.
They would then be intermingled with sensory precursors when they begin to
delaminate. Without clear evidence that this is the case, in
Fig. 6B we place the BP/LM
primordium beyond the neurogenic domain in the posterior part of the otic
cup.
Both shared and separate lineages give rise acoustic and vestibular ganglion neurons
It has been suggested that auditory and vestibular neuroblasts might arise
from separate developmental compartments in the medial versus lateral halves
of the ventral otocyst (Fekete and Wu,
2002). Gata3 expression in the medial half of the neurogenic
region (Lawoko-Kerali et al.,
2004
), combined with loss of the spiral ganglion but not the
vestibular ganglion in the Gata3-null
(Karis et al., 2001
), suggests
that the two ganglia may be derived from separate pools of progenitors in the
mouse otocyst. The majority of our neural lineage data supports this idea for
the chick ear: we estimate that 84% of ears with infected neurons contain
clones that colonize either acoustic or vestibular ganglia, but not both. On
the other hand, the segregation of the two pools must be incomplete, as we
estimate that the remaining 16% of ears with neural clones will contain a
clone that spans both ganglia. Such clones are generated from progenitors
uncommitted to auditory or vestibular ganglion fate at the time of infection.
We do not know how long these progenitors remain in the otic ectoderm after
infection, whether their progeny might be dispersing within the plane of the
epithelium, or when their progeny become committed relative to when they
delaminate. Nonetheless, the presence of clones that colonize both ganglia
suggests that the neurogenic region is not bisected by a strict lineage (or
compartment) boundary according to auditory versus vestibular fates, at least
up to stage 16, or
15 hours after the latest time (stage 14) at which
virus injection yielded bipotential clones.
Dispersion of clonally related cells in the otic epithelium
In the medial wall of utricle and in the endolymphatic apparatus, the sizes
of AP+ clusters are relatively small, typically fewer than five cells (data
not shown). In other regions of the inner ear, clusters typically contain a
larger number of cells that can be somewhat dispersed within a single
anatomical subdivision (Fig.
1C,E; data not shown). For example, some clones in the utricular
macula and the BP contain more than 50 scattered cells, although their
dispersion appears to be restricted by the borders formed with surrounding
non-sensory epithelium (e.g. Fig.
3D, Fig. 4A). In
the context of these regional differences in clone size, we note that the
dorsomedial wall of the otic vesicle, which generates the endolymphatic
apparatus, has substantially lower mitotic activity than the medial and
ventral walls at stage 16-28 (Lang et al.,
2000). Thus, regional differences in the proliferation potential
of otic progenitors may explain much of the variation in size and dispersion
of individual clones
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Conclusion |
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ACKNOWLEDGMENTS |
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