/ . Embryol. exp. Morph. Vol. 69, pp. 47-59, 1982
Printed in Great Britain © Company of Biologists Limited 1982
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Cell migration from the chick olfactory placode:
a light and electron microscopic study
By A. S. MENDOZA, 1 W. BREIPOHL 1 AND F. MIRAGALL 1
From the Institutjur Anatomie, Universitdtsklinikum Essen,
Bundesrepublik Deutschland
SUMMARY
The diflferentiation of the olfactory placode in the chick has been studied using light and
electron microscopy. Special attention was paid to the appearance of neuronal cells within
the placodal ectodermal thickening, the migration of cells out of this tissue and the
appearance of the first fila olfactoria in the differentiating olfactory mucosa.
Between the third and fifth day of incubation a large number of cells is observed leaving
the base of the invaginating olfactory placode, often in contact with thin axon bundles.
These cells are characterized by a well-developed Golgi apparatus, a considerable number
of mitochondria and dense-core vesicles. The morphology of these migrating cells resembles
that of cells observed near the basement membrane within the developing olfactory epithelium and is clearly different from the mesenchymal cells which are filled with polyribosomes. At the sixth day of incubation thick axon bundles can be observed within the
epithelium and the underlying lamina propria. The possible fate of the migrated epitheloid
cells is discussed.
INTRODUCTION
Cell migration from the olfactory placode was first described by His (1889)
and Kolliker (1890). Since these early investigations, cell migration from the
olfactory placode has been described for a variety of species: rat (Lejour,
1967), humans (O'Rahilly, 1967), rabbit (Filogamo & Robecchi, 1969), mole
(Milaire, 1974), chick (Robecchi, 1972; Milaire, 1974; Mendoza, 1979;
Laugwitz, 1980). Recently we have observed cell migration also from the
anlage of the accessory olfactory epithelium in the rat (Breipohl and coworkers, unpublished results). Little is known about the ultrastructure and
fate of these migrating cells. It is the aim of the present investigation therefore
to report some morphological observations of these cells in the early chick
embryo.
MATERIALS AND METHODS
HNL Nick chick eggs were incubated for 3, 3-5, 4, 4-5, 5-5 and 6 days at
38 °C. From the developing chicken embryos the olfactory epithelium was
examined both light- and electron microscopically. For each stage of development some 20 animals were used. The specimens were fixed by immersion in
1
Authors' address: Institut fur Anatomie, Universitatsklinikum Essen, Hufelandstr. 55,
4300 Essen 1, Federal Republic of Germany.
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A. S. MENDOZA, W. BREIPOHL AND F. MIRAGALL
Cell migration from the chick olfactory placode
49
a mixture of 3-2 % glutaralciehyde and 2-6 % paraformaldehyde in cacodylate
buffer (0-09 M, pH 7-35). The tissue samples were dissected free while immersed
in the fixative and kept in the same aldehyde solution another few hours at
4 °C. After fixation the tissue pieces were washed in Hank's solution, postfixed in 1 % osmium tetroxide (Dalton, 1955) for 2 h, washed and block
stained in dark glass bottles with 1 % uranyl acetate in sodium acetate buffer
(0-2 M, pH 5-0) for 30 min. The samples were then washed, dehydrated in
ethanol and embedded in Epon. Semi-thin sections (1 /im thick), as used for
light-microscopic study, were stained with 1 % Azure II-methylene blue. Thin
sections were picked up on Pioloform F-coated 200 mesh copper grids, contrasted according to Reynolds (1963) and investigated in a Philips 200 or 400
electron microscope.
To demonstrate the amount of extracellular material underneath the olfactory
placode in 4-day-old chick embryo the ruthenium red reaction was performed
according to the technique of Luft (1971a, b) and Mendoza (1980).
RESULTS
Light microscopy
Olfactory placodal thickening of the head ectoderm of chicken embryos
was first observed between 60 and 72 h of incubation at 38 °C. The maturing
sensory epithelium then showed a sharp and even demarcation from the underlying mesoderm (Fig. 1). From 72 h of incubation onwards a pronounced
irregular transition between the ectodermal and mesodermal tissue appeared.
This was caused by the appearance of cell groups at the epithelial base that
bulged towards the lamina propria. Between 72 h and 96 h of incubation,
groups of these cells migrated from the epithelium into the underlying mesenchyme (Fig. 2). Here they could be found till the beginning of the sixth day
of incubation, forming epitheloid islands or elongated cellular bands towards
the developing forebrain. Around the fifth day of incubation these cell groups
often lay in close association with the first outgrowing olfactory axons (Fig. 3).
Fig. 1. Olfactory placode (OP) from a 3-5-day-old chick embryo. Arrows indicate
the irregular contour of the epithelial base, x 590.
Fig. 2. Base of the invaginating olfactory placode (OP) from 5-day-old chick embryo.
Note groups of migrating cells (MC). Arrows indicate large intercellular spaces
communicating with the subjacent mesenchyme. x 590.
Fig. 3. Tangential section through the base of the invaginating olfactory placode
(OP) from a 4-5-day-old chick embryo. Note groups of migrating cells (MC) in
the subjacent mesenchyme in close association with developing axons (A), bv:
blood vessels, x 240.
Fig. 4, 5. Olfactory epithelium from the superior nasal concha of a 6-day-old
chick embryo. The epithelium is composed of five to six cell layers. Small asterisks
indicate thick axon bundles within the epithelium. Large asterisks indicate axon
bundles in the lamina propria. x480 and x 1200, respectively.
50
A. S. MENDOZA, W. BREIPOHL AND F. MIRAGALL
i .*i.**
Fig. <5. Base of the olfactory placode on the fourth day of incubation. Arrow
heads indicate basal lamina and extracellular fibrils. Arrows show mesenchymal
cell processes, x 15500.
Fig. 7. Base of the olfactory placode on the fourth day of incubation. Note strong
positive ruthenium red reaction in the region of the basal lamina, x 18500.
Cell migration from the chick olfactory placode
4
(jm
Fig. 8. Base of the olfactory placode from a 4-day-old chick embryo. Arrows indicate
cytoplasmic processes of the placodal cells, CP presumably processes of the
mesenchymal cells (Mes). x 12000.
Fig. 9. Base of the olfactory placode from a 3-5-day-old chick embryo. Note
group of migrating cells (MC). Arrow indicates a widened intercellular epithelial
space communicating with that in the underlying mesenchyme. x 4750.
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A. S. MENDOZA, W. BREIPOHL AND F. MIRAGALL
Fig. 10. Group of migrating cells (MC) from a 4-day-old chick embryo. Note the
well developed Golgi apparatus (G) and the presence of dense-core vesicles (arrow
heads). A: axons. x 15350.
Cell migration from the chick olfactory placode
Fig. 11. Migrated cells (MC). 4-5-day-old chick embryo. Arrow heads indicate
dense-core vesicles in the perikarya and in the cytoplasmic processes (P) filled
with polyribosomes. Arrows indicate dense-core vesicles in the axon-like processes
containing microtubules. G: Golgi apparatus, x 14700.
Fig. 12. Mesenchymal cell (Mes) and migrated cells (MC) in a 4-5-day-old chick
embryo. Note the large number of polyribosomes in the cytoplasm. Arrow heads
indicate dense-core vesicles of the migrating cells (MC). x 14000.
53
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A. S. MENDOZA, W. BREIPOHL AND F. MIRAGALL
13
Fig. 13. Base of the developing olfactory epithelium from a 5-5-day-old chick
embryo. Note the intraepithelial axon bundles (AB) which also contain abundant
dense-core vesicles (arrows). N: nucleus of epithelial cells, x 11350.
Cell migration from the chick olfactory placode
55
At the end of the sixth day of incubation, the outgrowing axons were more
numerous and arranged in thick bundles within the epithelial base and in the
lamina propria (Fig. 4). In the latter, they were ensheathed by thin, elongated
cells (Fig. 5). Typical epitheloid cell groups were no longer seen at this
developmental stage.
Electron microscopy
Electron microscopical investigation of the developing olfactory pit revealed,
up to the third day of incubation, a sharp demarcation from the underlying
lamina propria and a normal width of the intercellular space between the
basal feet of the epithelial cells. After this stage of development a more irregular
demarcation between both tissues became obvious. From 72 h of incubation
onwards growing numbers of fine processes and cell protrusions were observed
that bulged against the underlying mesodermal tissue (Fig. 8). In addition
to these, fine processes of mesenchymal cells were also observed in close
contact with the basal lamina of the developing olfactory epithelium (Figs. 6-8).
Both mesenchymal cell processes and the base of the developing olfactory
epithelium gave a positive ruthenium red reaction (Fig. 7). The reaction was
much stronger beneath the epithelial cells showing cytoplasmic processes and
extensive bulging. During further invagination of the olfactory placode the
intercellular cleft at the epithelial base was often enlarged and continued with
wide intercellular spaces in the subjacent mesenchyme (Figs. 8, 9). Groups of
epitheloid cells 'leaving' the epithelial base were characterized by a welldeveloped Golgi apparatus, considerable numbers of mitochondria and some
cisterns of rough endoplasmic reticulum (Figs. 9-11). The main characteristic
of the perikaryon of these cells was the presence of dense-core vesicles with
diameters between 100 and 200 nm (Fig. 11).
In the migrating cells two different cytoplasmic processes could be observed.
The first one was filled with polyribosomes and the second one lacked these
but contained many microtubules. In both these cell processes, dense-core
vesicles could be found (Fig. 11). The migrated epitheloid cells were similar
to some cells observed within the developing olfactory epithelium and clearly
different from the mesenchymal cells which were filled mainly with polyribosomes (Fig. 12). The axon bundles, which could be seen with the light
microscope by the sixth day of incubation, were characterized by the presence
of microtubules and mitochondria, by regularly occurring dense-core vesicles
and by the absence of ribosomes (Fig. 13).
DISCUSSION
The present investigation documents the differentiation of the olfactory pit
and groove in chick embryos between the third and sixth day of incubation.
Special attention has been paid to the fine structure of the epitheloid cells
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A. S. MENDOZA, W. BREIPOHL AND F. MIRAGALL
leaving the differentiating olfactory sensory epithelium, and to the appearance
of the first bundles of the fila olfactoria.
The ectodermal thickening occurring in olfactory placode formation is
caused by a pronounced local cellular proliferation. This local acceleration
of mitosis is accompanied by two morphological features: first, special apical
cell contacts and secondly, special morphological differentiation at the placodal
base (Mendoza, Miragall & Breipohl, 1980; Mendoza, 1980). The appearance
of gap junctions may play an important role in embryonic differentiation and
cell division (cf. Bennett, 1973; Loewenstein, 1973). In our opinion such a
hypothesis is also supported by the investigations of Decker & Friend (1974),
who found a temporary formation of gap junctions jn the course of amphibian
neurulation. Moreover a temporary appearance of these specialized cell contacts could be seen during olfactory placodal differentiation (Mendoza et ah
1980), and in the early stages of retinal development (Dixon & Cronly-Dillon,
1972). According to Fujisawa, Mori oka, Watanabe & Nakamura (1976)
'the fact that the number of cells with gap junctions attains a maximum at
the stage slightly before the cessation of cell proliferation may suggest that the
gap junction allows the passage of some factor(s) controlling the halt of cell
proliferation, which is followed by cell differentiation'. Another stimulus for
local ectodermal thickening may also result from developmental changes in
the underlying basal lamina.
Acid mucopolysaccharides and other acidic groups that are present in the
cell coat and in the basal lamina show a positive ruthenium red reaction
(Luft, 1971a, b; Blanquet, 1976a, b). As shown above the ruthenium red
reaction is less strong around the mesenchymal cell processes and scarcely
distributed fibrils deeper in the lamina propria than it is at the epithelial base.
We believe that this is due to the assembly of extracellular material forming
the underlying basal lamina (cf. Mendoza, 1980). The accumulation of extracellular ruthenium-red-positive material underneath the differentiating olfactory
placode is accompanied not only by enhanced epithelial mitosis but also by
a migration of cells out of the overlying epithelium. Investigating the process
of neurulation in chick embryo, Ebendal (1977) has assumed that the orientation
of the extracellular fibrils in the underlying mesenchyme apparently influences
the direction of migrating neural crest cells. Migration of epithelial cells is
influenced also by hyaluronate-containing extracellular matrix (Toole, 1973;
Pratt, Larsen & Johnston, 1975). A remarkably high content of hyaluronate
was shown by Ebendal (1977) in the mesenchyme surrounding the neural tube
of the chick embryo. With respect to the investigations of these authors we
argue therefore that the strong ruthenium red reaction that we obtained in the
region of the basal lamina of the developing olfactory placode is at least
partly due to an enhanced content of hyaluronate.
Migrating cells from the olfactory placode have been reported by different
authors (cf. Mendoza, 1979; Laugwitz, 1980). The nature and fate of these
Cell migration from the chick olfactory placode
57
remains to be discovered and more detailed fine structural investigation has
been missing so far. Filogamo & Robecchi (1969) have observed neuroblasts
in the olfactory pit of rabbits with a Koelle-positive reaction for acetylcholinesterase. Moreover they described Koelle-positive cells among the
developing fila olfactoria and they suggested that such cells were 'migratory
olfactory neuroblasts'. On the other hand Robecchi (1972) stated that at the
basal surface of the chick olfactory placode and in the underlying mesenchyme
along the outgrowing fila olfactoria, a nodular formation of cells appears that
are 'not of a nervous nature'. From the present description of the ultrastructural characteristics of these migrating cells, that shows them to be
similar to some cells observed within the developing olfactory epithelium, we
believe that they are migrating neurons. Whether all the observed axons within
such groups of migrating cells are fila olfactoria or have to be interpreted at
least partially as axons of the nervus terminalis (Brookover, 1914) requires
further investigation.
The thick bundles of olfactory fila seen in the base of the developing
olfactory epithelium and in the subjacent lamina propria from the sixth day
onwards were not seen in later developmental stages within the epithelium
(Mendoza, 1979). This fact may suggest that these bundles are formed primarily only by the grouping of developing axons from the sensory cells. Later
on they will be separated in the lamina propria by Schwann cells, probably
migrated from the neural crest, and within the epithelium by supporting cells.
Due to the observed close association of migrated epitheloid cells with the
outgrowing fila olfactoria it cannot be excluded, however, that Schwann cells
of the olfactory nerve may also originate from the olfactory placode. Such an
interpretation appears to agree with the investigations of O'Rahilly (1967) on
the developing olfactory nerve of man.
The dense-core vesicles found in the axon bundles as well as in placodal
and migrated cells have the typical appearance and diameter of norepinephric
granules described by Bloom & Fawcett (1975) in the adrenal medulla and
other paraneurons (Fujita, 1980). Cornwell-Jones & Marasco (1980) have
found a steroid-dependent content of norepinephrine in the olfactory bulb of
rats. Whether norepinephric granules do appear in neurons of the olfactory
bulb of the adult chicken as well and whether such granules may eventually
show an analogous behaviour as in the rat remains obscure, however.
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 114) and (Br.
358/5-1).
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(Received 20 July 1981, revised 14 December 1981)