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Development of the embryonic chick otic placode. II. Electron microscopic analysis

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Development of the Embryonic Chick Otic Placode
II. ELECTRON MICROSCOPIC ANALYSIS '
STEPHEN MEIER
Department of Anatomy, Uniuersity of Southern California School of Medicine,
2025 Zonal Avenue, Los Angeles, California 90033
ABSTRACT
The otic placode takes its origin from surface ectoderm. Prior
to the arrival of neural crest cells, surface epithelial cells adjacent to the neural
folds are squamous in shape and synthesize primarily interstitial bodies. However, by 26 hours of development, neural crest cells, using the undersurface of
the epithelium as a substratum, migrate away from the neural tube. Cells of
surface epithelium above the neural crest cells assume a columnar shape, and
the amount of intercellular space between adjacent epithelial cells is consequently reduced. Placode cells show extensive interdigitation apically as they
pseudostratify and invaginate, while it appears that many of the basal cells contribute components t o the underlying extracellular matrix. This extracellular
matrix interface between surface epithelium and neural crest cells is distinctly
fibrillar and less granular than that found between ordinary head ectoderm and
primary mesenchymal cells. Just prior to complete invagination as an otocyst,
otic placode cells in a region near the ventrolateral wall of the hindbrain extend
cell processes through discontinuities in the basal lamina and leave the otocyst.
These are likely to be the cells which contribute to the formation of the acoustico-facialis ganglion. These observations support the hypothesis that the development of the otic placode is the result of a tissue interaction between surface epithelium and neural crest cells.
The development of the otic placode has received little attention with the electron microscope even though early features of epithelial plate or placode formation have been studied in other systems. For instance, induction
of the neural plate and changes in epiblast cell
shape associated with development of the neural folds have been studied with the transmission electron microscope (TEM) (Gallera, '71;
Karfunkel, '74) as have mitotic activity and
interkinetic nuclear migration within the
wall of the neural tube (Sidman and Rakic,
'73; Seymour and Berry, '75). Using the scanning electron microscope (SEMI, several
workers have provided information related to
apical surface modifications of ectoderm during neurulation in birds (Bancroft and Bellairs, '74, '75; Bellairs and Bancroft, '75;
Jacob et al., '74; Revel, '74) and mammals
(Waterman, '75, '76) as well as during nasal
pit formation in the hamster (Waterman and
Meller, '73). The placodes of the chick embryo
(including otic) have also been reported to
ANAT. REC.
(1978)191: 459-478.
bear apical surface modifications (Bancroft
and Bellairs, '77). In addition, much attention
has been given to the extracellular matrix interface between the lens placode and optic
vesicle in chick embryos (Weiss and FittoJackson, '61; Hunt, '61; Silver and Wakely,
'74). The purpose of this electron microscopic
study was t o extend the initial light microscopic observations on otic development.
In the previous paper (Meier, '78), I reported
that the otic placode develops from the surface
epithelium of the cranial region concomitant
with the appearance of subjacent neural crest
cells (Meier, '77). In addition, i t was noted
that fibrillar extracellular matrix accumulated between neural crest cells and the otic
placode. By electron microscopy it is possible
to assess more critically the nature of the contact made between surface epithelium and
Received Oct. 25, ' 1 7 . Accepted Mar. 6, '78.
' This work was supported by G.R.S. Grant RR-05356 from the
University of Southern California School of Medicine and NIH Grant
DE-04613.
459
460
STEPHEN MEIER
neural crest cells and to evaluate the character and origin of the extracellular matrix
which lies between them. Although the focus
of this study is on tissue-interactions during
otic development, it should be appreciated
that these fine structural observations of placode formation are generally applicable t o epithelial morphogenesis. This same pattern of
development precedes the overt differentiation of all other organs of special sense, and
the central nervous system itself. (See review
of Hay and Meier, ’78.)
Rochester, New York) for 15 minutes a t room
temperature (Kelley et al., ’73), rinsed thoroughly again in buffer, post-fixed in 1%OsO,
a t room temperature for 60 minutes, and dehydrated through a series of graded alcohols.
Specimens were critical point dried using CO,
as the exchange fluid, mounted on aluminum
studs, and sputter-coated with 6-7 nm of goldpalladium alloy. Embryos were observed a t
20-25 kv with a JEOL JSM-35 scanning electron microscope.
MATERIALS AND METHODS
Apical surface morphology
At 26 hours of development, prior t o the
arrival of neural crest cells, the apical surface
of the head epithelium in the region where the
otic placode will develop, is rather unremarkable (fig. 1).As viewed with the scanning electron microscope, the polygonal cells usually
have one centrally located cilium, the rest
of the surface being smooth except a t the perimeter where adjacent epithelial cells meet.
Here the junctional zone is characterized by
the presence of broad microvilli which project
into the amniotic cavity, sometimes overlapping with the microvilli of adjacent cells.
The appearance of the apical surface of the
epithelium changes coincident with the arrival of the neural crest cells below (28 hours
of development). As the cells crowd within the
limits of the placode and invaginate (fig. 2),
they elongate and assume a columnar shape.
Thus the amount of free surface presented by
any one cell is greatly reduced. The cells
usually bulge out into the amniotic cavity,
their apical surfaces no longer smooth (fig. 3).
Instead, there are numerous, randomly distributed microvilli in addition t o the usual
central cilium. Most striking, however, is the
presence of long cytoplasmic extensions which
project from one cell and terminate on the apical surface of a non-adjacent cell. These elongate processes usually span several cells and
bear a prominent disc-shaped bulge midway
along their length (“A” in fig. 3). Although
they are not a common feature of non-placode
epithelium, they are occasionally present on
these cells as well. In addition, other long
processes of more uniform diameter and lacking the mid-bulge can be seen bridging non-adjacent placode cells. Several of these extensions may originate from a single cell.
Examination of thin sections of the apical
ends of otic placode cells with the transmission electron microscope shows considerable
Appropriately staged Rhode Island Red
chick embryos were obtained for processing
for electron microscopy as previously described (Meier, ’77). Embryos were fixed for 60
minutes on ice in a 2% glutaraldehyde-1%
osmium tetroxide mixture in 0.1 M cacodylate
buffer, pH 7.4 (Hasty and Hay, ’77). This mixture of fixatives was prepared fresh each time,
immediately before its anticipated use. Other
fixation procedures calling for the sequential
treatment of tissues first with aldehydes, then
with osmium, proved inadequate here. While
Karnovsky’s fixative (Karnovsky, ’65) or mixtures of aldehydes and acrolein (Luft, ’59) followed by treatment with osmium preserved
most embryonic tissues well, the apical surface of otic placodes, lens placodes, and neural
plate showed considerable blebbing, pitting,
and vesiculation. Consequently, the simultaneous aldehyde-osmium fixation procedure
was used here t o insure the preservation of
these rather unstable cell surfaces. For transmission electron microscopy, embryos were
stained en bloc for two hours with 0.5% Mg
uranyl acetate and embedded in Epon. Thin
sections were collected on carbon-supported
200-mesh grids, stained with uranyl acetate
followed by lead citrate (Reynolds, ’63) and examined with a JEOL 1OOC transmission electron microscope.
For scanning microscopy, embryos were
fixed by simultaneous aldehyde-osmium
treatment as described above. Some embryos
were rinsed in cacodylate buffer and transferred to clear Petri dishes where, under a dissecting microscope, they were cross sectioned
by a single stroke with a freshly cleaned razor
blade. Both halves of the sectioned embryos
were returned to buffer and processed with
unsectioned embryos as follows: Samples were
transferred t o a solution containing 0.5%thiocarbohydrazide (Eastman Organic Chemicals,
RESULTS
FINE STRUCTURE OF OTIC PLACODE
lateral interdigitation (fig. 4a). Adjacent cells
form junctional complexes that appear to be
mixtures of tight and intermediate junctions.
In favorable sections, portions of electron
dense regions are underlain with webs of
microfilaments. An interesting feature of the
apical cytoplasm is the presence of groups of
small membrane-bounded vesicles that are
located under the surface of the cell membrane above the level of the junctional complexes, a t sites where microvilli and other
cytoplasmic extensions project into the amniotic cavity (figs. 4a,b). Other membranebounded vesicles are located in the apical
cytoplasm adjacent to tight junctions (fig.
4a). As seen in figure 4c, vesicles of similar
size and appearance are clustered around that
portion of the Golgi apparatus nearest the apical end of the cells.
The chances of observing a cell process in its
entirety in any one section are remote since
they bend and extend for such long distances.
However, by meticulous search and examination of serial thin sections i t is possible to
trace long cytoplasmic extensions that span
non-adjacent epithelial cells. While some appear as isolated processes coursing above the
apical surface, others form junctional complexes where they contact neighboring cells.
Both kinds of processes usually contain microfilaments and membrane-bound vesicles, and
less frequently, microtubules, mitochondria
and profiles of rough endoplasmic reticulum
(fig. 4a). The area where processes bulge (as
seen in the SEMI appear t o be points of contact; each half of the process on either side of
the bulge belonging to a separate cell of origin.
Even though the apical surface of placode
cells may display artifactual membrane discontinuities, true syncytial bridges between
cells have not been observed.
Cellular ultrastructure
At 28 hours of development the head ectoderm consists of a single layer of typical epithelial cells, resting on a basal lamina (fig.
5a). The lateral surfaces of the cells are contiguous a t the apical region by virtue of their
junctional complexes, whereas basally, the
cells are adjoined by extensive interdigitation. However, much of the lateral surface between apical and basal regions borders intercellular space. Typically, the cytoplasm contains many free ribosomes and a few profiles
of rough endoplasmic reticulum and Golgi;
often, membrane-bounded coated vesicles are
461
seen preferentially located near the basal cell
surface. As non-placode epithelium continues
to differentiate, intercellular space is reduced,
adjacent cells being closely applied to one
another. While rough endoplasmic reticulum
and mitochondria are moderately abundant,
the most prominent cytoplasmic organelle is
the Golgi complex (fig. 5b). Located in the juxtanuclear cytoplasm, the Golgi region is extensive and has numerous small coated vesicles. Similar coated vesicles are often seen
along the basal cell surface (fig. 6) and these
vesicles contain electron dense material.
Cells located in the otic placode region, originally squamous to cuboidal and morphologically indistinguishable from adjacent, nonplacode cells, become more closely applied to
one another a t the expense of intercellular
space and pseudostratify. As the cells crowd
together and assume a tall columnar shape,
the cytoplasm becomes filled with long microtubules oriented parallel to the long axis of
the nucleus (fig. 7a). The cytoplasm also contains many free ribosomes and a moderate
amount of rough endoplasmic reticulum and
coated vesicles. The Golgi apparatus is perinuclear but usually assumes a lateral or apical
location. With the layering of placode cell nuclei, coated vesicles frequently can be seen
fused with the basal cell membrane. However,
i t is not uncommon to find similar coated vesicle-membrane confluency on the lateral surfaces of all cells included in the placode (fig.
7a, upper right triangular inset). Occasionally, intermediate junctions, desmosome-like
in appearance, can be seen along the lateral
surfaces of adjacent epithelial cells (fig. 7a,
rectangular inset). The cells at the base of the
placode rest on a continuous basal lamina (fig.
7b). However, by 46 hours of development, a
region of the placode a t the deepest point of invagination shows some signs of localized discontinuity of basal lamina. These spot breaks
in the basal lamina usually occur a t sites
where two or more placode cells meet. Epithelial cell processes, their cytoplasm containing
microfilaments, protrude into the extracellular space underneath the placode (fig. 7c).
Neural crest cells
The neural crest cells which leave the
rhombencephalon beginning at about 26 hours
of development are epithelial in origin but
exhibit mesenchymal behavior. Like mesenchyme, the cells leading the migration under
the surface epithelium extend long filopodia
462
STEPHEN MEIER
into the extracellular space ahead. Some cell and are not present a few hours later. Instead,
processes touch the basal lamina of the sur- as the cells develop an extensive Golgi comface epithelium (fig. 8 ) . However, the cell plex they are underlain by a granular submembranes of these two tissues have never stance that is more uniformly distributed
been observed t o form junctions with one along the basal surface outside the basal lamianother. Also, like mesenchyme cells, those na (fig. 6 and inset). Occasionally, collagen
neural crest cells that continue to spread un- fibers can be seen. The scanty distribution of
der the surface epithelium appear t o be active- fibers is verified by examination of the underly secretory. Their cytoplasm contains much surface of head ectoderm with the scanning
rough endoplasmic reticulum that has cister- electron microscope. Examination of the area
nal spaces distended with electron dense ma- indicated by the bold arrow in figure 10 shows
terial. The Golgi is well developed and is a few beaded fibers of various caliber coursing
usually located in juxtanuclear cytoplasm, over a background of granular material adherfacing one end of the cell. It is part of a cell ing to the basal lamina (fig. 11).
In contrast, the ECM which underlies the
center that includes a pair of centrioles and an
occasional developing cilium (fig. 9). Coated otic placode is distinctly fibrillar (fig. 7b).
vesicles, fused with the cell membrane, are Outside of the basal lamina, large diameter
also found frequently. Neural crest cells also collagen fibers, usually striated, are embedded
exhibit characteristics of epithelia, in that in granular material. The relative abundance
they are initially very close to one another and of collagen fibers can best be appreciated by
show macular-shaped junctions with fellow examination of the undersurface of a placode
neural crest cells (fig. 9, inset). Some neural where the neural crest cells have been physcrest cells even retain patches of basal lamina ically removed by a glancing blow with a razor
along the outer surface of the plasmalemma blade. As seen in figure 12, fibers are numerous and appear as an interconnecting, studded
(fig. 8, inset).
As the neural crest cells take up residence web superimposed on a background of coarser
under the future otic placode, extracellular granules. This pattern of extracellular matrix
matrix accumulates between them. Contact of distribution is found under almost every reneural crest cell filopodia with the basal lami- gion of the placode except where it closely apna of placode cells is observed less frequently. proaches the ventrolateral wall of the rhombCollagen fibers, distinctly striated, as well as encephalon. Here, the extracellular matrix is
smaller caliber unstriated fibers, accumulate much less fibrillar (fig. 7c). By 46 hours of dearound neural crest cell filopodia, as the cell velopment, this region of the otocyst is the site
soma moves farther away from the epithe- where some epithelial cells leave the placode
lium. Neural crest cells, or their processes, and enter the mesenchymal compartment.
underlie most regions of the placode, but they
DISCUSSION
are least prevalent under that part of the otoThe initial morphogenesis of the otic placcyst which most closely approaches the
rhombencephalon during invagination. For ode is similar to that reported for lens and
the duration of placode formation, fibers and neural plate. Surface epithelial cells crowd togranules accumulate in t h e extracellular gether a t the expense of intercellular space
space between the surface epithelium and the and elongate perpendicular to the basal lamineural crest cells. These matrix materials are na. Microtubules are oriented in the same diless prevalent a t the neural crest surface that rection and i t is likely that they mediate
changes in cell shape, and play a role in interfaces the primary mesenchyme.
kinetic nuclear migration (for review, see
Extracellular matrix (ECM)
Porter, '66). Soon, the cells of the otic placode
At 28 hours of development, the surface epi- pseudostratify and show evidence of regional
thelium is separated from the head mesen- organization; a t the free surface, the cells
chyme by a continuous basal lamina, about interdigitate, forming extensive junctional
70 nm thick. As reported by Low ('701, young complexes associated with microfilaments,
embryonic epithelium elaborates large masses while cell nuclei are located deeper in the
of extracellular matrix materials termed in- placode. A continuous basal lamina prevents
terstitial bodies (fig. 5 , inset). These spherical intimate contact of the plasma membranes of
entities, uniformly granular in appearance, placode cells and subjacent neural crest cells.
are transient features of surface epithelium While these are not unusual features for
FINE STRUCTURE O F OTIC PLACODE
placode formation, several novel aspects of
epithelial differentiation were also observed.
Observation of apical surface modifications
of t h e otic placode were facilitated by utilizing
a fixation procedure which prevents postfixation movement of membrane lipids (Hasty
and Hay, '77). The use of a fixative which simultaneously preserves protein and lipid moieties is crucial because of t h e extreme instability of t h e placode apical surface. Standard
sequential treatment of embryos first with
aldehydes, then with osmium, creates many
surface artifacts. Indeed, the "glass bell-like''
formation of developing neural tube reported
by Klika and Jelinek ('711, as well as t h e vesiculations and blebs observed by Ruggeri ('671,
Jacob et al. ('73) and Bancroft and Bellairs
('74) and t h e apical brush border reported for
human otic placode (O'Rahilly, '63) are quite
likely all artifacts of fixation.
In well preserved chick embryos, t h e most
prominent feature of t h e apical surface of otic
placode cells is t h e long cytoplasmic extensions which seem t o connect non-adjacent
cells. The majority of these projections a r e
0.1-0.2 p m in diameter and most closely
resemble microvilli, containing numerous microfilaments and sometimes extending 20 p m
i n length. While they occurred in about 5-7%
of non-placode cells, they were found on a s
many a s 20% of t h e surface placode cells. As
there a r e four times as many placode cells as
surface cells in equivalent areas of ectoderm,
i t is not surprising t h a t cytoplasmic extensions a r e observed more frequently in placode
regions. By examination of serial thin sections, these projections appear to be composed
of fused processes, each having arisen from a
different cell. Individual elongate projections
sprouting from a single cell were never observed. Bellairs and Bancroft ('75) identify
similar threads as midbodies, t h e remnant
connections between daughter cells after mitosis. The observation t h a t processes span
cells which a r e separated from each other by
various distances implies t h a t they represent
elongations of connections between previously
contiguous cells. Indeed, t h e presence of u n coated vesicles located under regions of cell
membrane which appear to be expanding, as
well as in cell processes themselves, suggests
t h a t they may contain material needed for
membrane assembly. Morphologically similar
vesicular profiles located in t h e Golgi region
further support t h e notion t h a t these vesicles
a r e destined t o contribute t o t h e formation of
463
new cell membrane. The structures noted here
are certainly similar, if not identical, to midbodies.
The junctional complexes, which outline t h e
elaborate interdigitation of adjacent epithelial cells, are probably composed of both tight
and intermediate junctions. In favorable sections, lateral membranes nearest the free surface show punctate contacts between their
outer leaflets, typical of tight junctions. I m mediately interior to these membrane specializations a r e intermediate junctions, which are
characterized by t h e presence of associated
broad bands of microfilaments. Other focal
membrane specializations t h a t resemble desmosomes were observed, but final verification
of their identity awaits t h e results of freeze
fracture experiments currently in a preliminary stage.
While morphogenesis of the surface epithelium proceeds toward invagination, the cytology of t h e underlying neural crest cells changes
very little. As they leave t h e neural tube, neural crest cells maintain cellular contact with
one another. While they extend filopodia t h a t
touch t h e basal lamina of the surface epithelium, cell junctions between crest cells and
placode cells were never seen. Indeed, t h e initial presence of t h e basal lamina and later the
additional buildup of extracellular matrix a t
this interface, actually serves to hold the neural crest cells a t some distance from the placode. With time, t h e cell bodies of neural crest
cells move farther away from the placode but
t h e two tissues remain intimately associated
with one another. It should be noted t h a t
while t h e otic placode takes its origin from
surface ectoderm originally located a t the
level of t h e first pharyngeal pouch, i t eventually comes to lie directly behind the first
pouch and invaginates into the body of the
second arch. However, neural crest cells subjacent to t h e otic placode remain associated
with i t throughout its displacement and invagination.
A unique feature of otic formation occurs
after about 45 hours of development. As the
placode invaginates, i t s ventromedian surface
closely approaches t h e hindbrain and neural
crest cells, uniformly present elsewhere under
t h e placode, a r e noticeably fewer in number
here. Also in this region, t h e basal lamina,
once continuous under the entire placode,
loses its integrity and epithelial cell processes
protrude into t h e underlying mesenchymal
compartment. These exiting epithelial cells
464
STEPHEN MEIER
are likely to be those that subsequently interact with neural crest cells and contribute to
the formation of the acoustico-facialis ganglion (Van Campenhout, '37a,b).
The extracellular matrix which accumulates between ordinary head ectoderm and
mesenchyme is morphologically distinct from
that which accumulates between the placode
and neural crest cells. Initially, the head ectoderm synthesizes interstitial bodies (Low, '70)
which appear as fuzzy masses of granules and
fine fibers adherent t o the epithelial basal
lamina. Soon, these entities become dispersed
in the subjacent extracellular space, sparsely
occupied by primary mesenchyme. The head
ectoderm, now replete with Golgi and coated
vesicles, as well as the mesenchyme, continues
to elaborate extracellular matrix that is predominantly granular, although a few fibers,
likely to be collagenous (Hay, '73), are observed. The matrix of the initial cell-free
space under the placode is also known to contain hyaluronate as well as glycoproteins
(Pratt et al., '74). However, the extracellular
matrix found between otic placode and neural
crest cells, is distinctly fibrillar. Long strands
of various caliber (5-25 nm) course between
these tissues. It seems likely that the extracellular matrix of the otic rudiment originates
from both placode and neural crest cells. The
placode cells have a prominent Golgi apparatus and numerous coated vesicles that can be
seen in the basal cytoplasm and fused with
lateral cell membranes. Likewise, neural crest
cells have a well developed Golgi region and
considerable amounts of rough endoplasmic
reticulum, often distended with flocculent
material. Coated vesicles fused with the cell
membrane are also common. Regardless of its
origin, the extracellular matrix that accumulates between neural crest cells and otic placode is more fibrillar than that found in nonplacode regions.
Finally, i t is tempting to speculate that the
development of the otic placode with the simultaneous appearance of subjacent neural
crest cells represents a developmentally significant tissue interaction. Second-order embryonic induction is emerging as a phenomenon
in which tissues of a dissimilar source promote, but do not necessarily initiate, each other's development across a n extracellular matrix interface (Hay and Meier, '78). During
otocyst development, the placode epithelium
and neural crest cells are separated by considerable amounts of extracellular matrix. In
addition, it is known t h a t the otocyst subsequently interacts with neural crest cells t o
form the bony labyrinth and acoustico-facialis
ganglion (Hamilton, '52; Van Campenhout,
'37a,b). However, the possibility that neural
crest cells may induce surface epithelial cells
to form the otic placode requires more direct
evidence that can only be obtained by methods
such as tissue separation and recombination.
ACKNOWLEDGMENTS
I am grateful t o Ms. Amy Erisman for her
excellent technical assistance as well as to
Doctors Douglas E. Kelly and Richard L. Wood
for their critical review of the manuscript.
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PLATE 1
EXPLANATION OF FIGURES
1 Scanning electron micrograph of surface of cranial epithelium at 26 hours of development. This image is also typical of t h e surface epithelium peripheral to the otic
placcde (similar area indicated by black arrow, fig. 2). X 3,800.
2 Scanning electron micrograph of cross sectioned otic placode a t 33 hours of development. Black arrow indicates area similar to that shown in figure 1, while white
arrow indicates area similar to that illustrated in figure 3. x 470.
3 Scanning electron micrograph of apical surface of otic placcde a t 33 hours of
development (area similar to that indicated by white arrow in fig. 2). Non-contiguous placode cells are joined by elongate cell processes, some bearing mid-length
bulges (A), while others are of more uniform diameter (B). X 10,800.
466
FINE STRUCTURE OF OTlC PLACODE
Stephen Meier
PLATE 1
467
PLATE 2
EXPLANATION OF FIGURES
Transmission electron micrographs of apical cytoplasm of otic placode cells at 33
hours of development.
4
a
Cells show considerable interdigitation and extensive junctional complexes. Small
membrane-bounded vesicles accumulate near t h e cell surface and in cell processes
(open arrows) and next to junctional complexes (dark arrows). X 164,300.
b Another area of placode apical surface membrane showing membrane-bounded vesicles accumulating under regions of cell extensions. X 17,000.
c
468
Region of Golgi apparatus facing apical cytoplasm, bearing numerous membranebounded vesicles. x 17,000.
FINE STRUCTURE OF OTIC PLACODE
Stephen Meier
PLATE 2
469
PLATE 3
EXPLANATION OF FIGURES
Transmission electron micrographs of surface epithelium, not participating in otic
placode formation.
5
a
Surface ectoderm a t 28 hours of development is composed of squamous shaped cells,
which have considerable intercellular space along their lateral surfaces (indicated
by a ). These cells contribute interstitial bodies to the extracellular space underlying t h e epithelium (inset) (Low, '70). X 7,200; inset, X 33,000.
b
By 46 hours of development, surface epithelial cells are more closely contiguous
along their lateral borders and Golgi lamellae are a conspicuous component of the
cytoplasm. x 10,100.
6
470
Transmission electron micrograph of basal surface of non-placode epithelium a t 46
hours of development (similar to t h a t illustrated in fig. 5b). The basal cytoplasm
bears coated vesicles and rests on a continuous basal lamina which separates it
from t h e predominantly granule-filled space. Occasionally, striated fibers (5-25 nm
in diameter) can be seen coursing in the granular background (inset). X 44,600;
inset, X 86,400.
FINE STRUCTURE OF OTIC PLACODE
Stevhen Meier
PLATE 3
47 1
PLATE 4
EXPLANATION OF FIGURES
7
Transmission electron micrographs of otic placode cells at 46 hours of development.
a
Placode cells are tall and columnar, and microtubules and the long axis of the
nucleus are oriented perpendicular to the basal surface. Intercellular space is reduced and lateral surfaces are often the sites of coated vesicle-membrane fusion
(triangular inset, upper right) as well as the site of macular junction formation
(rectangular inset, lower right). x 31,000; triangular inset, X 81,000.
b The basal placode cells usually rest on a continuous basal lamina which separates
the cells from the primarily fibrillar extracellular matrix. X 57,600.
c
472
By 46 hours of development, in a discrete region of t h e otic placode, epithelial cells
protrude from the otocyst through local discontinuities in the basal lamina. These
discontinuities usually occur a t sites where adjacent epithelial cells meet.
x 12,000.
FINE STRUCTURE OF OTlC PLACODE
Stephen Meier
PLATE 4
473
PLATE 5
EXPLANATION OF FIGURES
8 Transmission electron micrographs of neural crest cells that underlie the otic
placode a t 31 hours of development. Neural crest cell processes touch only the basal
lamina of the surface epithelium, and not the basal cell membrane itself. Neural
crest cells are actively synthetic, bearing considerable rough endoplasmic reticulum
and Golgi, cell membranes usually showing signs of fusion with coated vesicles. Occasionally, remnants of basal lamina (inset) adhere to neural crest cells, reflecting
their epithelial origin from the neural tube. X 20,200; inset, X 36,000.
9 Transmission electron micrographs of neural crest cells a t 33 hours of development.
Neural crest cells usually bear a cilium located near a cell center that includes a pair
of centrioles and extensive Golgi. They also form cell junctions with one another
when they are in close contact (inset). X 21,600; inset, X 79,200.
474
FINE STRUCTURE OF OTIC PLACODE
Stephen Meier
PLATE 5
475
PLATE 6
EXPLANATION OF FIGURES
10 Scanning electron micrograph of 46-hour-old embryo cross sectioned behind the second aortic arch. Viewed anteriorly, the otic placodes (now otocysts) invaginate into
the body of t h e second branchial arch. x 220.
11 Scanning electron micrograph of t h e undersurface of head ectoderm peripherally
adjacent to t h e developing otic placode from a region indicated by the black arrow
in figure 10. A few fibers of various caliber course over a granular background
which is likely to represent the basal lamina. X 14,800.
12 Scanning electron micrograph of the undersurface of the otocyst a t 46 hours of development from a region indicated by the black asterisk in figure 10. Numerous
fibers of various diameter, beaded in appearance, course over a finely granular
background. The extracellular matrix found here is considerably more fibrillar than
t h a t found under head ectoderm (compare with fig. 11).X 14,800.
476
FINE STRUCTURE OF OTlC PLACODE
Stephen Meier
PLATE 6
477
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development, embryonic, microscopy, placodes, chick, electro, analysis, otic
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