/. Embryol. cxp. Morph. Vol. 29, 3, pp. 549-558, 1973
549
Printed in Great Britain
A stereoscan study of the
origin of ciliated cells in the embryonic epidermis
of Ambystoma mexicanum
By F. S. BILLETT 1 AND T. H. COURTENAY 1
From the Department of Biology, The University of Southampton
SUMMARY
A stereoscan electron microscope has been used to survey the epidermis of axolotl embryos
as it becomes ciliated. The observations are consistent with much earlier ones that ciliated
cells first occur on the surface of the epidermis at about the time of the closure of the neural
folds. The cells are located first in the anterior dorsal region of the embryo at about the
one-somite stage. After this they rapidly increase in number and by the three- to six-somite
stage ciliated cells, which are isolated from one another, are scattered over the entire surface
of the embryo in numbers which approach those of much later stages (18 somites). At the
earlier stage, however, most of the ciliated cells lie below the general surface of the epidermis,
occupying pit-like depressions. This is in contrast to the later stage when they are raised
above the surface. The observations support the view that the precursors of the ciliated cells
lie beneath the outer epidermal layer of cells and that the ciliation of the embryonic surface
occurs when they move into the outer layer as they complete their differentiation.
INTRODUCTION
In amphibians the embryonic epidermis differentiates into two main types of
cell at a relatively early stage. The commonest cell type, which may be regarded
as typical of the embryonic and larval epidermis is mucus secreting (Pflugfelder
& Schubert, 1965). In Xenopus laevis, for instance (Billett & Gould, 1971), the
majority of cells are of this kind at the time that the embryos possess welldefined neural folds (approx. stage 17, Nieuwkoop & Faber, 1956). Ciliated
cells are also a feature of the embryonic epidermis. They differentiate at a
slightly later stage and in Xenopus are numerous by stage 20. Estimates from
1 /tm sections prepared for light microscopy indicate that about 1 in 10 of the
embryonic epidermal cells become ciliated (Billett, 1968).
Of particular interest is the origin of these cells; both light- and electronmicroscopic studies suggest that at an early stage of differentiation they lie
just beneath the surface of the epidermis (Billett, 1968; Steinman, 1968). Thus
in Xenopus laevis at stage 15, early neural folds, cells containing numerous
centrioles and areas of dense pre-centriolar material can be detected immediately
1
Authors' address: Department of Biology, Building 25, The University, Southampton,
SO9 5NH, U.K.
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F. S. BILLETT AND T. H. COURTENAY
below the outer epidermal cell layer (Billett & Gould, 1971). If we consider this
observation in relation to the appearance of ciliated cells on the surface of the
embryo a few stages later one is left with the impression that many ciliated cells
begin their differentiation beneath the surface of the epidermis before they appear,
in relatively large numbers, at the surface itself. The use of the scanning electron
microscope appears to confirm this general impression.
The observations reported here were made using axolotl embryos. In its
general features the development of the axolotl epidermis is very similar to that
of Xenopus and there is no reason to suppose that a stereoscan study of the
embryonic epidermis of Xenopus would not yield essentially the same results.
However, the larger cells of the urodele make the observations slightly easier.
A preliminary account of this work was given to the 22nd meeting of the British
Society for Developmental Biology in July 1971.
MATERIALS AND METHODS
Axolotl embryos were obtained as a result of the spontaneous mating of
mature individuals during the normal breeding season (New, 1966). The embryos
were staged using Harrison's table for Ambystoma maculatum as a guide (in
Hamburger, 1960). Selected stages were decapsulated using fine scissors and
forceps and immediately fixed in cold (5 °C) formaldehyde/glutaraldehyde
mixture buffered at pH 7-0 with 0-1 M sodium cacodylate (Karnovsky, 1965).
The embryos were fixed overnight, approx. 16 h, and then washed with the
cacodylate buffer. They were then completely dehydrated using one of the
following procedures.
The first method of dehydration involved freeze-substitution. Using a pipette
the embryos were removed from the sodium cacodylate solution, placed on
scoop-shaped pieces of aluminium foil about 1 cm square and immediately
plunged into a few ml of 1-methyl butane (iso-pentane) cooled in liquid nitrogen.
The methyl butane was contained in a 10 ml, glass-stoppered, Pyrex tube. The
boiling point of liquid nitrogen is some 50 °C below the melting point of
1-methyl butane, which becomes very viscous before it freezes. This means that
the plunging operation must be done fairly quickly. It must also be done very
carefully. Above all it should be remembered that methyl butane is very volatile
and forms explosive mixtures with air. It is essential to make sure that liquid
nitrogen is used as a coolant, not liquid air (Boyde & Wood, 1969). After the
freezing operation the embryos, in methyl butane, were placed in a deep freeze
at - 40 °C and 24 h later transferred to absolute ethanol at the same temperature. Approximately 3 weeks later the embryos were removed from the ethanol
and dried in a vacuum desiccator over phosphorus pentoxide.
The second dehydration procedure was to pass the fixed material through
a graded series of acetone-water mixtures, 25 %, 50 %, 75 % (v/v). The specimens
were left for 2 h in each mixture, and then for 3 h in absolute acetone followed
Ciliated cells in embryonic axolotl epidermis
551
by a second treatment with absolute acetone for about 16 h (overnight). Finally
the embryos were allowed to dry in air before storing in a vacuum desiccator
(Barber & Boyde, 1968; Fujita, Inoue & Kodama, 1968).
A day or two after the final stage of dehydration each specimen was attached
to the stereoscan stub with a small drop of silver paste (Johnson Matthey Metals,
London). Although the paste dries fairly quickly it is relatively easy to mount
the embryos in any desired orientation using watchmaker's forceps. In most
cases the embryos were placed on their right side so that the left side was fully
exposed and lay parallel to the stub surface.
Coating of the specimens was usually carried out 2 or 3 h after they had been
attached to the stereoscan stubs. Three methods of coating were used, namely
gold alone, carbon followed by gold and a gold-palladium mixture. The essential
details of the coating procedure are given by Barber & Boyde (1968).
After coating, the embryos were placed in a desiccator overnight and usually
observed in the stereoscan microscope (Cambridge, Mark 2 A) on the following
day. A beam voltage of 30 kV over a magnification range of x 40 to x 20000
was used.
Initially the embryos were examined at x 40 to x 60. This enabled the embryos
to be viewed as a whole and orientated in the desired direction for detailed
study. After orientation the lateral surface of the embryos were scanned systematically from head to tail using magnifications of up to 1000. Individual cells
were examined at x 2000 and details of cilia and surface protuberances at
x 10000 or x 20000. It is a fairly simple matter to pinpoint individual cells on
the embryonic surface. This is done by locating the chosen cell in the middle of
the viewing screen at high magnification (x 2000) and then, after photographing
the cell, reducing the magnification without moving the specimen until the
whole embryo or a recognizable part of it fills the screen. The chosen cell will,
of course, lie in the centre of any photograph taken at the low magnification.
The techniques were worked out and the original observations made on
larval stages of about 18 somites, a stage at which the epidermal surface is
strongly ciliated. An effort was then made to determine the stage at which
ciliated cells first appeared. These were obviously absent at the early neural-fold
stage, and equally obviously present at the three- to six-somite stages. Examination of the intervening stages, late neural folds to three somites, revealed the
presence of remarkably few ciliated cells. About 30 stereoscan preparations
were made of embryos ranging over the stages mentioned above. These embryos
were derived from six separate batches of eggs from different parents. Twenty
of these preparations were examined in detail; in many cases by scanning the
entire surface on one side of an embryo.
The results are presented in a developmental sequence. A brief description
of the epidermal surface at the stages just before ciliated cells appear is given
first. This is followed by accounts of the onset of ciliation and a description of
the fully ciliated epidermal surface of later stages.
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F. S. BILLETT AND T. H. COURTENAY
Ciliated cells in embryonic axolotl epidermis
553
The epidermal surface before ciliation
At low magnification (x 200) the embryonic epidermis at the neural-plate
and neural-fold stages, just before somites appear, presents a fairly uniform
field. The epidermal cells resemble rather flattish pebbles packed together to
form an uneven surface. Typically a 100 /tm square contains about 40 cells.
This density of packing is maintained over the entire surface of the epidermis,
although in the neural plate itself there appear to be a good many more cells
per unit area. Thus each epidermal cell occupies about 250 /*m2 of the total
surface which consists of approximately 15000 cells. At higher magnification
(x 4000) the cells appear to be separated by deep grooves which are bridged
occasionally by cytoplasmic processes. The cell surfaces are covered with small,
wart-like protuberances about 1 /.ivn in diameter, and on closer examination
( x 10000) the protuberances themselves are seen to bear even smaller rounded
projections. The surface appearance of the epidermal cells at these stages is very
similar to that of the non-ciliated cells seen at the slightly later stages. These
are shown at low magnification (Fig. 1 A) and at high magnification (Fig. 1F).
The development of ciliation
A few ciliated cells can be detected on the epidermal surface at about the
one-somite stage. These are located on the anterior dorsal surface of the embryo
near the centre of the region shown in Fig. 1 A. At this stage the cells are very
few in number. Despite a very thorough scan only two ciliated cells could be
detected on the entire left-hand side of the embryo. One of these cells can be
seen in Fig. 1 B. Relatively few cilia are borne on this cell and they are generally
confined to the central raised area of the cell surface. Apart from the few ciliated
cells the general appearance of the epidermal cells is very similar to that seen
in the slightly earlier stages. Individual cells are separated by deep clefts, they
FIGURE 1
Fig. 1. All specimens prepared by freeze-substitution using isopentane and liquid
nitrogen. C was coated with palladium-gold. The other specimens were coated
first with carbon and then with gold, cil., Cilia; c.c, ciliated cell; n.c, non-ciliated
epidermal cell; /?./, neural fold.
(A) The anterior dorsal region, about one quarter of the total embryonic surface
on that side, of an embryo at a neural fold - one somite stage. Most of the surface
is covered with typical (non-ciliated) epidermal cells, x 200.
(B) An isolated ciliated cell located in the centre of the area shown in A. x 2000.
(C) An axolotl embryo at about a three somite stage viewed from the dorsal
surface. A palladium-gold preparation, x 50.
(D) Ciliated cell and neighbouring epidermal cells from the anterior flank region
of a three somite embryo, x 2000.
(E) The same embryo as that shown in D. Ciliated cells and neighbouring
epidermal cells from posterior flank region, x 1000.
(F) The same embryo as that shown in D. Ciliated cells and non-ciliated neighbouring cells from posterior edge of embryo, x 1000.
36-2
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F. S. BILLETT AND T. H. COURTENAY
Ciliated cells in embryonic axolotl epidermis
555
are warty in appearance and the central region of the cell tends to bulge outwards. The entire epidermal surface is covered with about 16000 cells at a density
of some 35-40 cells per 100//m square.
As the somites develop, a dramatic change occurs on the epidermal surface.
At about the three-somite stage (Fig. 1C) large numbers of ciliated cells can be
detected. At the same time the epidermal cells in the anterior region of the
embryo become flattened, the clefts between them disappear and the cell
boundaries become demarcated by thin ridges (Fig. 1D). In the posterior region
of the embryo, however, the epidermal cells still resemble those of previous
stages (Figs. 1 E, F) but in addition numerous ciliated cells are seen. Typically
these lie slightly below the surface resembling shallow pits. In the tail region
these pits are much deeper, the ciliated cells appear to lie below the general
surface of the epidermis. The appearance of the cells in the posterior region
contrasts sharply with their appearance in the anterior flank region, where
they are at the same level and possess the same flattened profile as their nonciliated neighbours.
The degree of ciliation of individual cells varies, some are fully ciliated, but
in others (Fig. 1C) cilia are confined to a central area on the cell surface. The
entire epidermal surface consists of approximately 18000 cells of which about
a tenth are ciliated. In the posterior region there are about 35-40 cells per 100 /*m
square, whereas in the anterior region there are approximately 30 cells in the
same unit area.
The fully ciliated epidermal surface
The fully ciliated condition is seen in Fig. 2, showing photographs of various
aspects of an 18-somite embryo. A low-power scan (Figs. 2 A, B) reveals that
the epidermal surface contains many ciliated cells which are apparently located
at random. The chief features are the fact that they are always isolated from
one another and fairly evenly distributed although there are significantly more
cells in the upper half of the embryo than the lower half. The cells are obviously
raised above the general level of the epidermal surface, resembling numerous
FIGURE 2
Fig. 2. All specimens coated with carbon followed by gold. A-E, Frozen in isopentane-liquid nitrogen; F, acetone preparation, cil., Cilia, c.c, ciliated cell, ext.g.,
rudiment of external gill filament; opt. v., position of optic vesicle.
(A) Head region of an axolotl embryo of about 18 somites, x 100.
(B) Trunk region of embryo shown in A. x 60.
(C) A ciliated cell surrounded by typical epidermal cells from the centre of the
region shown in B. x 1000.
(D) A partially ciliated cell with a central bald area from the trunk region of the
embryo shown in B. x 2000.
(E) Individual cilia and adjacent microvilli of a ciliated cell (from embryo shown
in B). x 10000.
(F) Individual cilia from an acetone preparation. Note that the microvilli are
poorly denned, x 10000.
556
F. S. BILLETT AND T. H. COURTENAY
small blisters. Higher magnification (Fig. 2C) shows that the neighbouring
epidermal cells are flattened and bordered by narrow ridges. This condition
exists over the entire surface in contrast to the earlier somite stages. As before,
the degree of ciliation varies. Most of the cells are fully ciliated (Fig. 2C) but
some have bald areas in the centre of the cell surface (Fig. 2D). Details of the
ciliated surfaces are shown in Figs. 2E and F. Many of the ciliary filaments are
apparently fused together in groups of two or three shortly after they emerge
from the cell surface. Numerous microvilli are interspersed with the cilia. From
the early to the late somite stages (at least up to 18 somites) the total number of
epidermal cells does not appear to rise significantly. A rough estimate of the
number of epidermal cells in the specimen shown in Fig. 2 comes to 18000 and,
as before, about 1 in 10 of the cells is ciliated.
DISCUSSION
The work reported here and the recently published observations of Tarin
(1971) using earlier stages of Xenopus demonstrate the usefulness of the stereoscan electron microscope for the study of the surface structure of amphibian
embryos. The machine also obviously provides a spectacular means of viewing
whole embryos in three dimensions. A number of technical problems were
encountered during the work and it would seem helpful at this point to mention
them briefly. The fixation of the embryos for stereoscan work presents no real
difficulties. The frequently used Karnovsky mixture (formaldehyde/glutaraldehyde in cacodylate buffer) produced good preparations. The use of osmium,
either as a primary fixative or for post-fixation, does not seem suitable. Osmium
makes the embryos brittle and leads to an excessively cracked surface. Coating
becomes difficult and surface charging occurs in the microscope. In most of
our specimens a few surface cracks appeared but more care with the fixation
process might avoid the shrinkage and deformation which leads to cracking
(Fujita, Tokunaga & Inoue, 1971). Of the two methods of dehydration described
in this paper there is no doubt that the longer, more tedious, freeze-substitution
technique is superior to the more straightforward acetone drying. Thus, at
high magnifications the acetone preparations lack the surface detail seen in the
freeze-dried material. (Compare Figs. 2E and F). For attaching the specimens
a silver paste is superior to double-sided adhesive tape for the obvious reason
that good conductivity between the coated specimen and the stereoscan stub
is assured. Both carbon followed by gold and a palladium-gold mixture proved
superior to gold alone for coating purposes.
In their general appearance the ciliated cells of the embryonic epidermis of
the axolotl resembles those of similar cells seen in other tissues (Barber & Boyde,
1968). The individual cilia possess the usual dimensions (see Sleigh, 1962). The
apparent fusion of small groups of cilia at their distal ends (Figs. 2E and F) is
almost certainly an artifact of the method of preparation. However, the fine
Ciliated cells in embryonic axolotl epidermis
557
detail of these cells and of the neighbouring non-ciliated epidermal cells is not
the concern of this article. Sufficient information for our main purpose is provided by the gross appearance of the cells in relation to the development of the
embryo as a whole.
The onset of ciliation in the amphibian embryo is of course very readily
observed, and it is generally agreed that this occurs at about the neural-fold
stage (Assheton, 1896). Ambystoma species are no exception to this rule {A.
mcxicanum, Woerdmann, 1925; A. punctatum, Twitty, 1928). However, it must
be remembered that the actual time at which cilia develop on the epidermal
surface is not strictly correlated with stage for, as Twitty (1928) observed, a low
temperature favours early development of cilia relative to stage. Bearing this
in mind, however, the detection of a few (probably less than ten) ciliated cells
by the stereoscan at about the one-somite stage of A. mexicanum correlates well
with previous, and far older, observations. The location of the first cilia reported
in this paper is also in accord with Twitty's work, roughly corresponding to his
'small area on the outer cranial portion of the neural fold'. Again the raised
profile of the ciliated cells of the later somite stages was also observed by Twitty,
although it is in fact extremely difficult to view the living surface of the embryo
at high magnification. What is so striking about the stereoscan work, compared
with previous observations, is that it reveals just how quickly the epidermal
surface becomes peppered with ciliated cells which are apparently isolated from
one another. Thus from about ten cells at the one-somite stage the number
rises sharply to an almost full complement of cells (probably in the region of
2000) by the time the embryo has added a few more somites. The ciliated cells
appear on the surface in a broadly antero-posterior order, indicating a gradient
of cell differentiation spreading from an original focus in the upper anterior
flank region. Associated with this gradient, but not necessarily causally connected with it, is a flattening of the epidermal cells on the surface of the embryo.
Apart from the suddenness with which the ciliated cells appear the most
interesting point is the way in which they apparently emerge from beneath the
general surface of the epidermis. At the three-somite stage the epidermis,
particularly in the posterior half, has many pit-like depressions, both shallow
and deep, and each pit appears to correspond to a single ciliated cell. The
possibility that these pits are artifacts produced by the fixation or subsequent
procedures has been considered. However, it must be borne in mind that at the
above-mentioned stage a single embryo can be seen to possess not only the
pit-like depressions on the posterior epidermal surface but also ciliated cells
of normal appearance and surface dimension in the anterior region. Only if
the posterior and anterior ends of the embryo behave differently during the
preparative procedures can the possibility of an artifact be entertained. We are
fairly confident that the stereoscan preparations do not grossly distort the
appearance of the living epidermis. Thus both the rapidity with which the cells
appear at a certain stage of development and their general relation to the
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F. S. BILLETT AND T. H. COURTENAY
neighbouring non-ciliated cells at this stage is consistent with the idea that the
ciliation of the amphibian epidermis is caused by the movement of precursor
cells, lying immediately beneath the surface, the cells becoming ciliated as they
move outwards to become part of the outer epidermal layer.
We are indebted to our colleagues in the Engineering Department for training us in the
use of the stereoscan and to Dr L. G. E. Bell of the Department of Biology for some helpful
discussion about the techniques employed.
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(Received 25 August 1972, revised 19 October 1972)