/. Embryo/, exp. Morph. Vol. 59, pp. 249-261, 1980
Printed in Great Britain © Company of Biologists Limited 1980
249
Cellular morphology in haploid amphibian embryos
By MARK S. ELLINGER 1 AND JUDITH A. MURPHY 1
From the Department of Zoology, and Center for Electron Microscopy,
Southern Illinois University at Carbondale
SUMMARY
External surfaces of haploid and diploid embryos of Bombina orientalis were examined
with the scanning electron microscope to determine the possible contribution of cellular
morphology to the amphibian haploid syndrome. Cellular anomalies were prevalent in all
surface areas of haploid embryos. The epithelium appeared uneven due to the displacement
of ciliated cells and the rounded apical surfaces of the non-ciliated cells. The ratio of ciliated
to non-ciliated cells was altered in comparison to diploid embryos. Cells of the gill filaments
and adhesive organs were abnormal in morphology, and the adhesive organs themselves
were fused into a single large rudiment in haploid embryos. Uniformity of cell size was
markedly reduced in head regions of haploid embryos with severe microcephaly. Haploid
and diploid embryos elaborated mucoid matrices over the surface cells when removed from
the fertilization envelope.
It is apparent that aberrant cellular morphologies are widespread in haploid embryos,
and it is likely that these defects are major contributors to the gross morphological anomalies
of the haploid syndrome.
INTRODUCTION
Haploid amphibians have been examined frequently as models of altered
cellular and nucleo-cytoplasmic interactions in teratogenesis (Porter, 1939;
Hertwig, 1913; Subtelny, 1958; Hamilton, 1966; Briggs, 1949; Graham, 1966;
Dasgupta & Matsumoto, 1972; Hronowski, Gillespie & Armstrong, 1979).
Haploid embryos typically exhibit a 'haploid syndrome', including changes
in body proportions, edema, and poorly developed circulation. Most die
before feeding, though some haploid embryos have proceeded to more advanced
stages (Fankhauser, 1945; Miyada, 1977; Hronowski et al. 1979). The reasons
for haploid developmental anomalies have been difficult to determine. One
possibility is that deleterious recessive genes, normally masked in diploid
embryos, are expressed during haploid development (Darlington, 1937). This
has been examined in several ways, one of the most revealing of which has
been the production of homozygous diploid embryos by nuclear transplantation.
Subtelny (1958) reported that such embryos, in which recessive alleles should
be expressed, displayed better development than haploids to the late tailbud stage; thereafter, deficiencies became apparent and none of the larvae
1
Authors' address: Department of Zoology, and Center for Electron Microscopy, Southern
Illinois University at Carbondale, Carbondale, Illinois 62901, U.S.A.
250
M. S. ELLINGER AND J. A. MURPHY
metamorphosed. He concluded that abnormal early development of haploids
was due to the haploid condition itself, while deleterious recessive alleles (or
some other nuclear condition) affected only later embryonic stages.
Hertwig (1913) suggested that an altered nucleo-cytoplasmic volume ratio
was the basis of the haploid syndrome. This was tested by Briggs (1949), who
produced androgenetic haploid Rana pipiens embryos from eggs of varying
sizes. He observed a reduction in the severity of abnormalities in haploids
derived from small eggs, though the major aspects of the syndrome were still
expressed. He concluded, like Subtelny. that the major haploid anomalies were
due to the haploid state itself.
Regulation of cell number has also been examined to test the importance
of altered nucleo-cytoplasmic ratio. Graham (1966) discovered that haploid
Xenopus laevis embryos achieved a normal nucleo-cytoplasmic ratio by undergoing an additional cleavage between 4 and 12 h postfertilization. Hronowski
et al. (1979) observed a similar phenomenon in haploid axolotl embryos, in
which normal nucleo-cytoplasmic ratios were attained by the 32-cell stage.
Hamilton (1966) and Dasgupta & Matsumoto (1972) have suggested that
haploid embryos, due to a lack of heterozygosity, are unable to adjust to
developmental stresses imposed by variations in the external environment or
by variations in quality of egg cytoplasm.
The level of organization at which haploid inviability is expressed has not
yet been determined. The deficiencies are not necessarily cell lethal, since
parabiosis of haploid and diploid Pleurodeles waltii embryos greatly prolongs
the survival of the haploids (Gallien & Beetschen, 1960; Gallien, 1963, 1967),
and since production of haploid/diploid chimeras also enhances the viability
of the haploid cells (Hamilton, 1963). However, it is possible that intrinsicallydeficient haploid cells under these conditions were supplied with compensatory
materials from neighbouring diploid cells. It has also been demonstrated that
haploid Rana pipiens cells can be maintained in vitro as stable cell lines (Freed
& Mezger-Freed, 1970), indicating again that the haploid condition is not, per
se, cell lethal. However, the ability of haploid cells to multiply and maintain
themselves in vitro says little about their ability to participate in normal
embryogenesis and differentiation. Mezger-Freed (1975) suggested that those
genes coding for ubiquitous proteins (necessary for maintenance in vitro) may
be present as multiple copies in a haploid genome, while those coding for
proteins characteristic of differentiated cells may be present as fewer or single
copies. Thus, haploid cells might be capable of surviving in vitro but still be
deficient in the ability to undergo normal differentiation.
We have examined the morphology of cells in the external epithelium of
diploid Bombina orientalis embryos, using scanning electron microscopy (SEM)
(Ellinger & Murphy, 1979). We felt that it would be useful to extend these
observations to haploid embryos in order to more fully characterize the cellular
contribution to the haploid syndrome.
Cellular morphology in haploid amphibian embryos
251
MATERIALS AND METHODS
The Bombina orientalis used in this study were from a colony maintained
in the Department of Zoology, Southern Illinois University at Carbondale. The
colony was initiated in 1973 with adults purchased from Dr George Nace, The
Amphibian Facility, University of Michigan, Ann Arbor, Michigan, U.S.A.
Ovulation, spermiation and amplexus were induced by injection of 2501.U.
human chorionic gonadotropin (Sigma) beneath the dorsal skin. Fertilized
eggs were reared at 20-22 °C in ringer bowls containing 10 % Barth's Solution
X (Barth & Barth, 1959). Embryos were staged according to Sussman & Betz
(1978).
For production of haploids, freshly-inseminated eggs were observed (50 x )
until the appearance of the 'black dot' (Porter, 1939; Carlson & Ellinger,
1980), marking the location of the second meiotic spindle. The black dot
(including maternal chromosomes) was then destroyed in situ by ruby laser
microbeam-induced coagulation (McKinnell, Mims & Reed, 1969; Ellinger,
King & McKinnell, 1975). The development of such eggs is guided solely by
the haploid set of paternal chromosomes. The androgenetic haploids were
reared in parallel with laser-irradiated diploids from the same matings. Ploidy
was confirmed by observations of cell size and by chromosome preparations
of the tail epithelium (DiBerardino, 1962).
Prior to preparation for EM, approximately 125 haploid embryos were
pre-screened, using a dissecting microscope, to select for those specimens with
'typical' features of the haploid syndrome (similar to the specimen shown in
Fig. 1 b). Unless otherwise indicated, haploid embryos displaying gross edema,
acephaly, pronounced microcephaly, extreme dorsal-ventral flexure, or other
severe abnormalities, were not used in this study. The following developmental
stages were examined: stage 14-16 (neural fold to neural tube) - 19 embryos;
stage 17 (tail bud)-nine embryos; stage 18 (muscular response) - eight
embryos; stage 19-20 (gill filaments) - 2 0 embryos; stage 21 (open mouth)12 embryos. Over 100 diploid embryos of similar stages were also examined
with SEM.
For SEM, embryos were removed from the fertilization envelope and jelly
coats with watchmakers forceps, placed immediately in 3 % glutaraldehyde in
0-1 M cacodylate buffer for 3 h, washed, then post-fixed in Parducz fixative
(six parts 2 % OsO4 to one part saturated HgCl2), or 1 % OsO4, for 20 min.
Fixed samples were dehydrated through a graded series of ethanol (25, 50, 75,
95, 100 %), placed in Freon TF, then critical-point dried in a Bomar 900 EX
critical-point dryer. Specimens were mounted on Cambridge-type stubs with
silver paint, coated with carbon and PdAu, and examined in a Cambridge
Mark IIA SEM operated at 20 kV.
For transmission electron microscopy (TEM), samples were fixed in 3 %
glutaraldehyde in 0-1 M cacodylate at 4 °C for 4 h, buffer-washed three times,
252
M. S. ELLINGER AND J. A. M U R P H Y
Fig. 1. Comparison of diploid and haploid B. orientalis embryos. (A) Diploid gillbud embryo (stage 19-20). (B) Haploid embryo, stage 19-20, 'typical' haploid
syndrome. Note differences in body proportions of the haploid embryo. Scale
line equals 500 /tm.
then post-fixed in 1 % aqueous OsO4 for \\ h. Samples were dehydrated in
two changes each of a graded series (25, 50, 75, 95, 100 %) of ethanol, and
finally twice in propylene oxide for 15 min and embedded in Epon 812 according
to Luft (1961). Sections were cut with a diamond knife on a Reichert 0mU2
ultramicrotome and stained with lead citrate (Venable & Coggeshall, 1965)
and uranyl acetate. Sections were viewed in an Hitachi HUl 1 AB TEM operated
at 50 kV or a JEOL 100C operated at 60 kV.
Cellular morphology in haploid amphibian embryos
253
Fig. 2. (A) Surface cells in the flank region of a stage-18 (muscular response)
embryo. Exocytotic apertures are visible on the non-ciliated cells. Scale line
equals 10 /tm. (B) (C) TEM of exocytotic vesicles and apertures. Same age as
(A). Mitochondria are most numerous near the exocytotic loci. Scale lines equal
1 /tm.
17
EMB59
254
M. S. ELLINGER AND J. A. MURPHY
Cellular morphology in haploid amphibian embryos
255
Table 1. Number of ciliated and non-ciliated cells
Embryo (stage)
(1)
(2)
(3)
(4)
(5)
(6)
Haploid (20)
Haploid (20)
Diploid (20)
Diploid (20)
Diploid (18)
Diploid (18)
No. of
ciliated
cells
No. of
non-ciliated
cells
Ciliated/nonciliated
85
59
117
61
105
496
377
259
224
127
219
997
0-23
0-23
0-52
0-48
0-48
0-49
RESULTS
The most obvious morphological abnormality in haploid B. orientalis
embryos was a change in body proportions (Fig. 1), as has been reported in
other amphibians. The features reported below represent perceptions derived
from observations of 68 haploid embryos. Though minor differences were
seen, no gross deviations from the typical morphologies shown in Figs. 1-12
were observed in the numerous embryos examined.
Two major cell types were present in the epidermis of diploid B. orientalis
embryos - ciliated and non-ciliated (Ellinger & Murphy, 1979). By stage 18
(muscular response), non-ciliated cells displayed varying numbers of surface
apertures which we believe to be manifestations of exocytotic events (Fig. 2a-c).
By stage 19-20 (gill bud), the apical surfaces of many non-ciliated cells became
almost entirely occupied by exocytotic apertures (Fig. 3). Both ciliated and
non-ciliated cells in diploid embryos possessed relatively flat external surfaces
(Fig. 3).
Ciliated and non-ciliated cells were also present in the haploid epidermis.
However, apical cell surfaces were frequently not flat, but rounded; ciliated
FIGURES
3-7
Fig. 3. Surface cells in the flank region of a stage-19 to -20 embryo. Note the
abundance of exocytotic apertures. Scale line equals 10/tm.
Fig. 4. Flank region of haploid embryo shown in Fig. 1 (B). The epithelium is
uneven, with many ciliated cells appearing to have partially emerged from the
surface. Compare with diploid epithelium in Figs. 2(A), 3.
Fig. 5. 'Wrinkled' epithelium in belly region of a haploid embryo, stage 19-20.
Scale line equals 50 /tm.
Fig. 6. Epithelium in flank region of haploid embryo stage 19-20. Exocytosis is not
as prominent or uniform as in diploid embryos of the same age. (see Fig. 3) Scale
line equals 10 /im.
Fig. 7. TEM of haploid epithelial cell, stage 19-20. Secretory vesicles are numerous
at the cell apex, though vesicle surface rupture appears reduced in comparison to
diploids. Scale line equals 1 /*m.
17-2
256
M. S. ELLINGER AND J. A. MURPHY
Cellular morphology in haploid amphibian embryos
257
Fig. 13. Epithelium in head region of a haploid embryo with severe microcephaly.
Note non-uniformity of cell size. Scale line equals 10/tm.
Fig. 14. Mucoid matrix on the surface of haploid epithelial cells. Unlike previous
embryos which werefixedimmediately upon removal from the fertilization envelope,
this embryo was hatched 24 h before fixation. Diploid embryos elaborate similar
mucous coats if removed from the fertilization envelope 24 h prior to fixation. Scale
line equals 1 /tm.
cells often appeared to have emerged partially from the plane of the surrounding
non-ciliated cells (Fig. 4), giving the epidermis an uneven appearance. Gross
epithelial wrinkling was observed in the belly regions of several haploid embryos
(Fig. 5). Exocytotic apertures were seen on haploid non-ciliated cells, though
the extent of exocytosis remained quite variable between embryos and between
FIGURES
8-12
Fig. 8. Head region of haploid embryo with gross morphological appearance
similar to the embryo shown in Fig. 1 (B). The ventral adhesive organs are fused
into one large structure (arrows). Note unevenness of the surrounding head
epithelium. Scale line equals 500/tm.
Fig. 9. SEM of cells in the haploid ventral adhesive organ. In a similar area of the
diploid adhesive organ, all the cells would be heavily ciliated. Arrows indicate
examples of probable rudimentary or immature cilia. Scale line equals 5 /im.
Fig. 10. SEM of diploid gill filaments. (A) Low magnification showing gross
morphology. Scale line equals 100 fim. (B) Higher magnification of a cell located
on one of the filaments in (A). A porous matrix obscures the underlying surface
features. Cilia from an adjacent cell are visible on the right. Scale line equals
1 /im.
Fig. 11. Gill filaments of a haploid embryo. Note unevenness of the surface
epithelium. Scale line equals 50 /im.
Fig. 12. Higher magnification of gill filament cells shown in Fig. 11. Note the
raised margins of the exocytotic apertures and the absence of the porous matrix
as seen in Fig. 10(B). Scale line equals 5 fim.
258
M. S. ELLINGER AND J. A. MURPHY
cells of the same embryo (Fig. 6). TEM observations suggested that numbers
of exocytotic vesicles were comparable to diploid cells, but that the frequency
of vesicle rupture at the surface was reduced (Fig. 7).
With SEM, relative numbers of ciliated and non-ciliated cells in the flank
regions were compared in sibling diploid and haploid embryos. At stage 20, the
ratio of ciliated/non-ciliated cells for diploids was 0-51 (529 cells scored in
two embryos), and for haploids was 0-23 (780 cells scored in two embryos).
Since haploid embryos are often regarded as developmentally retarded in
comparison to diploids, we also checked the ratio in stage-18 diploid embryos.
The observed ratio of 0-49 (1817 cells scored in two embryos) at this stage
was not significantly different from that observed at stage 20 (Table 1).
The ventral adhesive organs in diploid embryos first appeared (stage 16-17)
as widely separated, paired rudiments. By stage 18, the rudiments had moved
toward the ventral midline and, in many cases, had become partially fused in
the posterior regions. By stage 20, the organs were again separate, with normal
epithelium between them (Ellinger & Murphy, 1979). In all haploid embryos
examined, the two adhesive organs remained fused, and the extent of fusion
was often much greater than that seen in diploids (Fig. 8). All of the cells
within most areas of the diploid adhesive organs became ciliated. Many of the
haploid adhesive organ cells failed to become fully ciliated, displaying instead
rudimentary and/or immature cilia (Fig. 9).
Diploid gill filaments appeared as finger-like projections covered with ciliated
and non-ciliated cells (Fig. 10a). The apical surfaces of these cells were coated
with a finely-porous matrix of unknown composition and function (Fig. 10b).
The haploid gill filament epithelium was relatively uneven, and the filaments
themselves were typically shorter and thicker than their diploid counterparts
(Fig. 11). We did not observe a porous matrix over the haploid gill filament
cells; many of the non-ciliated cells displayed unusual exocytotic apertures
with raised margins (Fig. 12).
The above abnormalities were observed on embryos with 'typical' haploid
syndromes (similar to that shown in Fig. 1). We also examined the head regions
of several haploid embryos with severe microcephaly. The surface contours
were extremely uneven, and there was a pronounced non-uniformity of cell
size, due mainly to the presence of overly-large cells (Fig. 13).
Diploid B. orientalis embryos, when removed from the fertilization envelope
and jelly coats and exposed directly to 10 % Barth's Solution X for 12-24 h,
accumulate a prominent mucoid matrix over the surface epithelial cells (Ellinger
& Murphy, 1979). We found that haploid embryos, under the same conditions,
were able to elaborate a similar mucoid matrix (Fig. 14).
Cellular morphology in haploid amphibian embryos
259
DISCUSSION
While haploid abnormalities have been described in detail at the organ/
tissue level (Porter, 1939; Briggs, 1949; Subtelny, 1958; Miyada, I960), few
studies have examined haploid embryos from the standpoint of cellular
morphology. Ellinger (1979) described melanophore patterns in haploid and
diploid embryos of B. orientalis. Diploid epidermal melanophores, after
exhibiting a number of specific cell-cell interactions, organized into a distinctive
orthogonal network throughout the tadpole. Haploid melanophores were
altered in morphology, failed to exhibit the cell-cell interactions seen in diploid
embryos, and remained randomly oriented. While haploid embryos had
approximately twice as many epithelial cells, on a unit-area basis, as diploid
embryos, numbers of melanophores were five to six times greater in haploid
embryos.
Our present study provides additional evidence that haploid embryos possess
inherent defects at the cellular level. Indeed, the component cells of the entire
external surface (gill filaments, adhesive organs, and the rest of the epidermis)
were found to be affected. The observed anomalies were comparable to those
of the haploid melanophores in two major respects. First, cellular morphologies
were abnormal. Second, as with the melanophore/epithelial cell ratio, the
ratio of ciliated/non-ciliated cells was altered in haploid embryos. While it
has been suggested that a haploid embryo as a whole possesses twice as many
cells on a unit-area basis (Hamilton, 1963), the present results suggest that
cell numbers are not regulated uniformly in haploid embryos. This must be
considered as at least a partial basis for genesis of the haploid syndrome. It
remains to be determined whether the altered ratio in this case is due to a
paucity of ciliated cell precursors, an overabundance of non-ciliated cells, or
the failure of presumptive ciliated cells to become ciliated. The latter possibility
is suggested by the fact that cilia on many presumptive ciliated cells of haploid
adhesive organs were absent or rudimentary.
Previously, we suggested that the exocytotic apertures seen on diploid
embryos represent loci of mucous secretion, but that the secreted products
remain soluble as long as the embryo remains within the fertilization envelope
(Ellinger & Murphy, 1979). Thus, we did not observe a mucous coat unless
the embryo was removed from the jelly coats and fertilization envelope and
exposed directly to the external medium for a number of hours prior to fixation.
In spite of the somewhat irregular appearance of exocytotic apertures on
haploid cells, the embryos were nevertheless able to elaborate mucous-like
surface matrices indistinguishable, with SEM, from diploid mucous coats. It
is therefore unlikely that haploid embryos suffer from a deficiency in mucoid
production or secretion.
The surface matrix on diploid gill filament cells, which appears while the
embryo is yet within the fertilization envelope, was not observed on haploid
260
M. S. ELLINGER AND J. A. MURPHY
gill filament cells. It is possible that the absence of such a matrix, coupled
with the general foreshortening of the filaments themselves, results in suboptimal respiratory functions in haploid embryos. This might contribute to
the poor circulation and edema that are characteristic of the haploid syndrome.
However, gill filament defects cannot be the primary basis of the syndrome,
since haploid anomalies are apparent prior to the formation of the gill filaments
(Subtelny, 1958; Hamilton, 1966).
Defects in the haploid adhesive organs were manifested in two ways. First,
gross morphology was altered in that the rudiments failed to separate as they
did in diploid embryos. Further, the extent of fusion was usually much greater
than in the diploids, such that the area appeared as a single large rudiment.
Second, cellular features within the haploid adhesive organs were also altered.
While we have not carried out an extensive TEM analysis, it appears from
SEM observations that the proportion of ciliated cells in the diploid B. orientalis
adhesive organ (Ellinger & Murphy, 1979) is much greater than in either
Rana pipiens (Kessel et al. 1974) or Xenopus laevis (Picard & Gilloteaux, 1976).
The failure of many haploid adhesive organ cells to become fully ciliated
could be due to a number of variables. One possibility would be defects in
microtubule structure or assembly. As microtubules are likely major contributors
to cell morphology (Stephens & Edds, 1976), such defects might also explain
the structural anomalies in other haploid cells.
This study represents the first analysis of haploid amphibian embryos with
SEM. The observations strongly suggest that defects at the cellular level are
widely disseminated in haploid B. orientalis embryos. While it is conceivable
that these defects derive secondarily from a primary defect in a specific tissue
or organ rudiment, it is more likely that the cellular anomalies themselves
are a major contributing feature to the development of the haploid syndrome.
This work was, in part, supported by grant no. 79-3 from the American Cancer Society,
Illinois Division. The Center for Electron Microscopy of SIU-C is acknowledged for use
of its equipment, partially purchased with Biomedical Sciences support grant no. FR-1 S05
FR07118-01.
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{Received 4 January 1980, revised 10 March 1980)