cellular morphology in haploid amphibian...

/ . Embryo/, exp. Morph. Vol. 59, pp. 249-261, 1980 2 4 9Printed in Great Britain © Company of Biologists Limited 1980

Cellular morphology in haploid amphibian embryos

By MARK S. ELLINGER1 AND JUDITH A. MURPHY1

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 examinedwith the scanning electron microscope to determine the possible contribution of cellularmorphology to the amphibian haploid syndrome. Cellular anomalies were prevalent in allsurface areas of haploid embryos. The epithelium appeared uneven due to the displacementof ciliated cells and the rounded apical surfaces of the non-ciliated cells. The ratio of ciliatedto non-ciliated cells was altered in comparison to diploid embryos. Cells of the gill filamentsand adhesive organs were abnormal in morphology, and the adhesive organs themselveswere fused into a single large rudiment in haploid embryos. Uniformity of cell size wasmarkedly reduced in head regions of haploid embryos with severe microcephaly. Haploidand diploid embryos elaborated mucoid matrices over the surface cells when removed fromthe 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 anomaliesof the haploid syndrome.

INTRODUCTION

Haploid amphibians have been examined frequently as models of alteredcellular 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 changesin body proportions, edema, and poorly developed circulation. Most diebefore feeding, though some haploid embryos have proceeded to more advancedstages (Fankhauser, 1945; Miyada, 1977; Hronowski et al. 1979). The reasonsfor haploid developmental anomalies have been difficult to determine. Onepossibility is that deleterious recessive genes, normally masked in diploidembryos, are expressed during haploid development (Darlington, 1937). Thishas been examined in several ways, one of the most revealing of which hasbeen the production of homozygous diploid embryos by nuclear transplantation.Subtelny (1958) reported that such embryos, in which recessive alleles shouldbe expressed, displayed better development than haploids to the late tail-bud stage; thereafter, deficiencies became apparent and none of the larvae

1 Authors' address: Department of Zoology, and Center for Electron Microscopy, SouthernIllinois 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 haploidswas due to the haploid condition itself, while deleterious recessive alleles (orsome other nuclear condition) affected only later embryonic stages.

Hertwig (1913) suggested that an altered nucleo-cytoplasmic volume ratiowas the basis of the haploid syndrome. This was tested by Briggs (1949), whoproduced androgenetic haploid Rana pipiens embryos from eggs of varyingsizes. He observed a reduction in the severity of abnormalities in haploidsderived from small eggs, though the major aspects of the syndrome were stillexpressed. He concluded, like Subtelny. that the major haploid anomalies weredue to the haploid state itself.

Regulation of cell number has also been examined to test the importanceof altered nucleo-cytoplasmic ratio. Graham (1966) discovered that haploidXenopus laevis embryos achieved a normal nucleo-cytoplasmic ratio by under-going an additional cleavage between 4 and 12 h postfertilization. Hronowskiet al. (1979) observed a similar phenomenon in haploid axolotl embryos, inwhich normal nucleo-cytoplasmic ratios were attained by the 32-cell stage.

Hamilton (1966) and Dasgupta & Matsumoto (1972) have suggested thathaploid embryos, due to a lack of heterozygosity, are unable to adjust todevelopmental stresses imposed by variations in the external environment orby variations in quality of egg cytoplasm.

The level of organization at which haploid inviability is expressed has notyet been determined. The deficiencies are not necessarily cell lethal, sinceparabiosis of haploid and diploid Pleurodeles waltii embryos greatly prolongsthe survival of the haploids (Gallien & Beetschen, 1960; Gallien, 1963, 1967),and since production of haploid/diploid chimeras also enhances the viabilityof the haploid cells (Hamilton, 1963). However, it is possible that intrinsically-deficient haploid cells under these conditions were supplied with compensatorymaterials from neighbouring diploid cells. It has also been demonstrated thathaploid Rana pipiens cells can be maintained in vitro as stable cell lines (Freed& Mezger-Freed, 1970), indicating again that the haploid condition is not, perse, cell lethal. However, the ability of haploid cells to multiply and maintainthemselves in vitro says little about their ability to participate in normalembryogenesis and differentiation. Mezger-Freed (1975) suggested that thosegenes coding for ubiquitous proteins (necessary for maintenance in vitro) maybe present as multiple copies in a haploid genome, while those coding forproteins characteristic of differentiated cells may be present as fewer or singlecopies. Thus, haploid cells might be capable of surviving in vitro but still bedeficient in the ability to undergo normal differentiation.

We have examined the morphology of cells in the external epithelium ofdiploid Bombina orientalis embryos, using scanning electron microscopy (SEM)(Ellinger & Murphy, 1979). We felt that it would be useful to extend theseobservations to haploid embryos in order to more fully characterize the cellularcontribution 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 maintainedin the Department of Zoology, Southern Illinois University at Carbondale. Thecolony was initiated in 1973 with adults purchased from Dr George Nace, TheAmphibian 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. Fertilizedeggs were reared at 20-22 °C in ringer bowls containing 10 % Barth's SolutionX (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 lasermicrobeam-induced coagulation (McKinnell, Mims & Reed, 1969; Ellinger,King & McKinnell, 1975). The development of such eggs is guided solely bythe haploid set of paternal chromosomes. The androgenetic haploids werereared in parallel with laser-irradiated diploids from the same matings. Ploidywas confirmed by observations of cell size and by chromosome preparationsof the tail epithelium (DiBerardino, 1962).

Prior to preparation for EM, approximately 125 haploid embryos werepre-screened, using a dissecting microscope, to select for those specimens with'typical' features of the haploid syndrome (similar to the specimen shown inFig. 1 b). Unless otherwise indicated, haploid embryos displaying gross edema,acephaly, pronounced microcephaly, extreme dorsal-ventral flexure, or othersevere abnormalities, were not used in this study. The following developmentalstages were examined: stage 14-16 (neural fold to neural tube) - 19 embryos;stage 17 (tail bud)-nine embryos; stage 18 (muscular response) - eightembryos; stage 19-20 (gill filaments) - 2 0 embryos; stage 21 (open mouth)-12 embryos. Over 100 diploid embryos of similar stages were also examinedwith SEM.

For SEM, embryos were removed from the fertilization envelope and jellycoats with watchmakers forceps, placed immediately in 3 % glutaraldehyde in0-1 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 EXcritical-point dryer. Specimens were mounted on Cambridge-type stubs withsilver paint, coated with carbon and PdAu, and examined in a CambridgeMark 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,


Fig. 1. Comparison of diploid and haploid B. orientalis embryos. (A) Diploid gill-bud embryo (stage 19-20). (B) Haploid embryo, stage 19-20, 'typical' haploidsyndrome. Note differences in body proportions of the haploid embryo. Scaleline equals 500 /tm.

then post-fixed in 1 % aqueous OsO4 for \\ h. Samples were dehydrated intwo changes each of a graded series (25, 50, 75, 95, 100 %) of ethanol, andfinally twice in propylene oxide for 15 min and embedded in Epon 812 accordingto Luft (1961). Sections were cut with a diamond knife on a Reichert 0mU2ultramicrotome and stained with lead citrate (Venable & Coggeshall, 1965)and uranyl acetate. Sections were viewed in an Hitachi HUl 1 AB TEM operatedat 50 kV or a JEOL 100C operated at 60 kV.


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 lineequals 10 /tm. (B) (C) TEM of exocytotic vesicles and apertures. Same age as(A). Mitochondria are most numerous near the exocytotic loci. Scale lines equal1 /tm.

17 EMB59


Table 1. Number of ciliated and non-ciliated cells

Embryo (stage)

(1) Haploid (20)(2) Haploid (20)(3) Diploid (20)(4) Diploid (20)(5) Diploid (18)(6) Diploid (18)

No. ofciliatedcells

8559

11761

105496

No. ofnon-ciliated

cells

377259224127219997

Ciliated/non-ciliated

0-230-230-520-480-480-49

RESULTS

The most obvious morphological abnormality in haploid B. orientalisembryos was a change in body proportions (Fig. 1), as has been reported inother amphibians. The features reported below represent perceptions derivedfrom observations of 68 haploid embryos. Though minor differences wereseen, no gross deviations from the typical morphologies shown in Figs. 1-12were observed in the numerous embryos examined.

Two major cell types were present in the epidermis of diploid B. orientalisembryos - ciliated and non-ciliated (Ellinger & Murphy, 1979). By stage 18(muscular response), non-ciliated cells displayed varying numbers of surfaceapertures 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 becamealmost entirely occupied by exocytotic apertures (Fig. 3). Both ciliated andnon-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

F I G U R E S 3-7

Fig. 3. Surface cells in the flank region of a stage-19 to -20 embryo. Note theabundance of exocytotic apertures. Scale line equals 10/tm.

Fig. 4. Flank region of haploid embryo shown in Fig. 1 (B). The epithelium isuneven, with many ciliated cells appearing to have partially emerged from thesurface. 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 notas prominent or uniform as in diploid embryos of the same age. (see Fig. 3) Scaleline equals 10 /im.

Fig. 7. TEM of haploid epithelial cell, stage 19-20. Secretory vesicles are numerousat the cell apex, though vesicle surface rupture appears reduced in comparison todiploids. Scale line equals 1 /*m.

17-2


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 previousembryos which were fixed immediately upon removal from the fertilization envelope,this embryo was hatched 24 h before fixation. Diploid embryos elaborate similarmucous coats if removed from the fertilization envelope 24 h prior to fixation. Scaleline equals 1 /tm.

cells often appeared to have emerged partially from the plane of the surroundingnon-ciliated cells (Fig. 4), giving the epidermis an uneven appearance. Grossepithelial wrinkling was observed in the belly regions of several haploid embryos(Fig. 5). Exocytotic apertures were seen on haploid non-ciliated cells, thoughthe extent of exocytosis remained quite variable between embryos and between

FIGURES 8-12

Fig. 8. Head region of haploid embryo with gross morphological appearancesimilar to the embryo shown in Fig. 1 (B). The ventral adhesive organs are fusedinto one large structure (arrows). Note unevenness of the surrounding headepithelium. Scale line equals 500/tm.Fig. 9. SEM of cells in the haploid ventral adhesive organ. In a similar area of thediploid adhesive organ, all the cells would be heavily ciliated. Arrows indicateexamples of probable rudimentary or immature cilia. Scale line equals 5 /im.Fig. 10. SEM of diploid gill filaments. (A) Low magnification showing grossmorphology. Scale line equals 100 fim. (B) Higher magnification of a cell locatedon one of the filaments in (A). A porous matrix obscures the underlying surfacefeatures. Cilia from an adjacent cell are visible on the right. Scale line equals1 /im.Fig. 11. Gill filaments of a haploid embryo. Note unevenness of the surfaceepithelium. Scale line equals 50 /im.Fig. 12. Higher magnification of gill filament cells shown in Fig. 11. Note theraised margins of the exocytotic apertures and the absence of the porous matrixas seen in Fig. 10(B). Scale line equals 5 fim.


cells of the same embryo (Fig. 6). TEM observations suggested that numbersof exocytotic vesicles were comparable to diploid cells, but that the frequencyof vesicle rupture at the surface was reduced (Fig. 7).

With SEM, relative numbers of ciliated and non-ciliated cells in the flankregions were compared in sibling diploid and haploid embryos. At stage 20, theratio of ciliated/non-ciliated cells for diploids was 0-51 (529 cells scored intwo embryos), and for haploids was 0-23 (780 cells scored in two embryos).Since haploid embryos are often regarded as developmentally retarded incomparison 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 stagewas 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 movedtoward the ventral midline and, in many cases, had become partially fused inthe posterior regions. By stage 20, the organs were again separate, with normalepithelium between them (Ellinger & Murphy, 1979). In all haploid embryosexamined, the two adhesive organs remained fused, and the extent of fusionwas often much greater than that seen in diploids (Fig. 8). All of the cellswithin most areas of the diploid adhesive organs became ciliated. Many of thehaploid adhesive organ cells failed to become fully ciliated, displaying insteadrudimentary and/or immature cilia (Fig. 9).

Diploid gill filaments appeared as finger-like projections covered with ciliatedand non-ciliated cells (Fig. 10a). The apical surfaces of these cells were coatedwith a finely-porous matrix of unknown composition and function (Fig. 10b).The haploid gill filament epithelium was relatively uneven, and the filamentsthemselves were typically shorter and thicker than their diploid counterparts(Fig. 11). We did not observe a porous matrix over the haploid gill filamentcells; many of the non-ciliated cells displayed unusual exocytotic apertureswith raised margins (Fig. 12).

The above abnormalities were observed on embryos with 'typical' haploidsyndromes (similar to that shown in Fig. 1). We also examined the head regionsof several haploid embryos with severe microcephaly. The surface contourswere extremely uneven, and there was a pronounced non-uniformity of cellsize, due mainly to the presence of overly-large cells (Fig. 13).

Diploid B. orientalis embryos, when removed from the fertilization envelopeand 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).


DISCUSSION

While haploid abnormalities have been described in detail at the organ/tissue level (Porter, 1939; Briggs, 1949; Subtelny, 1958; Miyada, I960), fewstudies have examined haploid embryos from the standpoint of cellularmorphology. Ellinger (1979) described melanophore patterns in haploid anddiploid embryos of B. orientalis. Diploid epidermal melanophores, afterexhibiting a number of specific cell-cell interactions, organized into a distinctiveorthogonal network throughout the tadpole. Haploid melanophores werealtered in morphology, failed to exhibit the cell-cell interactions seen in diploidembryos, and remained randomly oriented. While haploid embryos hadapproximately twice as many epithelial cells, on a unit-area basis, as diploidembryos, numbers of melanophores were five to six times greater in haploidembryos.

Our present study provides additional evidence that haploid embryos possessinherent defects at the cellular level. Indeed, the component cells of the entireexternal surface (gill filaments, adhesive organs, and the rest of the epidermis)were found to be affected. The observed anomalies were comparable to thoseof the haploid melanophores in two major respects. First, cellular morphologieswere abnormal. Second, as with the melanophore/epithelial cell ratio, theratio of ciliated/non-ciliated cells was altered in haploid embryos. While ithas been suggested that a haploid embryo as a whole possesses twice as manycells on a unit-area basis (Hamilton, 1963), the present results suggest thatcell numbers are not regulated uniformly in haploid embryos. This must beconsidered as at least a partial basis for genesis of the haploid syndrome. Itremains to be determined whether the altered ratio in this case is due to apaucity of ciliated cell precursors, an overabundance of non-ciliated cells, orthe failure of presumptive ciliated cells to become ciliated. The latter possibilityis suggested by the fact that cilia on many presumptive ciliated cells of haploidadhesive organs were absent or rudimentary.

Previously, we suggested that the exocytotic apertures seen on diploidembryos represent loci of mucous secretion, but that the secreted productsremain soluble as long as the embryo remains within the fertilization envelope(Ellinger & Murphy, 1979). Thus, we did not observe a mucous coat unlessthe embryo was removed from the jelly coats and fertilization envelope andexposed directly to the external medium for a number of hours prior to fixation.In spite of the somewhat irregular appearance of exocytotic apertures onhaploid cells, the embryos were nevertheless able to elaborate mucous-likesurface matrices indistinguishable, with SEM, from diploid mucous coats. Itis therefore unlikely that haploid embryos suffer from a deficiency in mucoidproduction or secretion.

The surface matrix on diploid gill filament cells, which appears while theembryo is yet within the fertilization envelope, was not observed on haploid


gill filament cells. It is possible that the absence of such a matrix, coupledwith the general foreshortening of the filaments themselves, results in sub-optimal respiratory functions in haploid embryos. This might contribute tothe 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 theydid in diploid embryos. Further, the extent of fusion was usually much greaterthan 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 fromSEM observations that the proportion of ciliated cells in the diploid B. orientalisadhesive organ (Ellinger & Murphy, 1979) is much greater than in eitherRana pipiens (Kessel et al. 1974) or Xenopus laevis (Picard & Gilloteaux, 1976).The failure of many haploid adhesive organ cells to become fully ciliatedcould be due to a number of variables. One possibility would be defects inmicrotubule structure or assembly. As microtubules are likely major contributorsto cell morphology (Stephens & Edds, 1976), such defects might also explainthe structural anomalies in other haploid cells.

This study represents the first analysis of haploid amphibian embryos withSEM. The observations strongly suggest that defects at the cellular level arewidely disseminated in haploid B. orientalis embryos. While it is conceivablethat these defects derive secondarily from a primary defect in a specific tissueor organ rudiment, it is more likely that the cellular anomalies themselvesare 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 useof its equipment, partially purchased with Biomedical Sciences support grant no. FR-1 S05FR07118-01.

REFERENCES

BARTH, L. G. & BARTH, L. J. (1959). Differentiation of cells of the Rana pipiens gastrula inunconditioned medium. J. Embryoi. exp. Morph. 2, 210-222.

BRIGGS, R. (1949). The influence of egg volume on the development of haploid and diploidembryos of the frog. Rana pipiens. J. exp. Zool. I l l , 255-294.

CARLSON, J. T. & ELLINGER, M. S. (1980). The reproductive biology of Bombina orientalis.Herp. Rev. 11, 11-12.

DARLINGTON, C D . (1937). Recent Advances in Cytology, 2nd ed., ch. xi. Philadelphia:Blakiston.

DASGUPTA, S. & MATSUMOTO, L. (1972). The haploid syndrome in isogenic haploid frogembryos of Rana pipiens derived by nuclear transplantation. / . exp. Zool. 180, 413-420.

DIBERARDINO, M. A. (1962). The karyotype of Rana pipiens and investigation of its stabilityduring embryonic differentiation. Devi Biol. 5, 101-126.

Cellular morphology in haploid amphibian embryos 261ELLINGER, M. S. (1979). Ontogeny of melanophore patterns in haploid and diploid embryos

of the frog, Bombina orientalis. J. Morph. 162, 77-92.ELLINGER, M. S., KING, D. R. & MCKINNELL, R. G. (1975). Androgenetic haploid develop-

ment produced by ruby laser irradiation of anuran ova. Radiat. Res. 62, 117-122.ELLINGER, M. S. & MURPHY, J. A. (1979). Embryo surface morphology during post-gastrula

development of the frog, Bombina orientalis, as revealed by scanning electron microscopy./ . Anat. 129, 361-376.

FANKHAUSER, G. (1945). The effects of changes in chromosome number on amphibiandevelopment. Q. Rev. Biol. 20, 20-78.

FREED, J. J. & MEZGER-FREED, L. (1970). Stable haploid cultured cell lines from frogembryos. Proc. natn. Acad. Sci., U.S.A. 65, 337-344.

GALLIEN, L. (1963). Differenciation sexuelle d'individus haploides du triton Pleurodeleswaltii M1CAH mis en parabiose: Effects de competition dans les associations ? 2N:<?1N.C. r. hebd. Seanc. Acad. Sci., Paris 257, 2890-2893.

GALLIEN, L. (1967). Development d' individus haploides adultes eleves en parabiose chezle triton Pleurodeles waltii MICAH: Syndrome de l'haploidie et differenciation sexuelle./ . Embryol. exp. Morph. 18, 401-426.

GALLIEN, L. & BEETSCHEN, J. C. (1960). Differenciation sexuelle et gametogenese abortivechez un male haploide d'Urodele {Pleurodeles waltii), eleve en parabiose. C. r. hebd.Seanc. Acad. Sci., Paris 251, 1655-1657.

GRAHAM, C. F. (1966). The effect of cell size and DNA content on the cellular regulationof DNA synthesis in haploid and diploid embryos. Exp I Cell Res. 43, 13-19.

HAMILTON, L. (1963). An experimental analysis of the development of the haploid syndromein embryos of Xenopus laevis. J. Embryol. exp. Morph. 11, 267-278.

HAMILTON, L. (1966). The role of the genome in the development of the haploid syndromein Anura. / . Embryol. exp. Morph. 16, 559-568.

HERTWIG, G. (19.13). Parthogenesis bei wirbeltieren, hervorgerufen durch artfremdenradiumbestrahlten Samen. Arch. mikr. Anat. 81, II, 87-128.

HRONOWSKI, L., GILLESPIE, L. L. & ARMSTRONG, J. B. (1979). Development and survival ofhaploids of the Mexican axolotl, Ambystoma mexicanum. J. exp. Zool. 209, 41-48.

KESSEL, R. G., BEAMS, W. & SHIH, C. Y. (1974). The origin, distribution and disappearanceof surface cilia during embryonic development of Rana pipiens as revealed by scanningelectron microscopy. Am. J. Anat. 141, 341-360.

LUFT, J. H. (1961). Improvements in epoxy resin embedding methods. J. Biophys. Biochem.Cytol. 9, 409-414.

MCKINNELL, R. G., MIMS, M. F. & REED, L. A. (1969). Laser ablation of maternalchromosomes in eggs of Rana pipiens. Z. Zellforsch. mikrosk. Anat. 93, 30-35.

MEZGER-FREED, L. (1975). Mutagenesis of haploid cultured frog cells. Genetics 79, 359—372.

MIYADA, S. (1960). Studies on haploid frogs. J. Sci. Hiroshima Univ. B, Div. 1, 19, 1-56.MIYADA, S. (1977). A three-year-old haploid frog. Sci. Rep. Lab. Amph. Biol., Hiroshima

Univ. 2, 213-227.PICARD, J. J. & GILLOTEAUX, J. (1976). Scanning electron microscopy of Xenopus laevis

cement gland. Archs Biol., Bruxelles 87, 401-414.PORTER, R. R. (1939). Androgenetic development of the egg of Rana pipiens. Biol. Bull.

mar. biol. Lab., Woods Hole 77, 233-257.STEPHENS, R. E. & EDDS, K. T. (1976). Microtubules: structure, chemistry, and function.

Physiol. Rev. 56, 709-777.SUBTELNY, S. (1958). The development of haploid and homozygous diploid frog embryos

obtained from transplantation of haploid nuclei. / . exp. Zool. 139, 263-305.SUSSMAN, P. & BETZ, T. W. (1978). Embryonic stages: morphology, timing, and variance

in the toad Bombina orientalis. Can. J. Zool. 56, 1540-1545.VENABLE, J. H. & COGGESHALL, R. (1965). A simplified lead citrate stain for use in electron

microscopy. / . Cell Biol. 25, 407-408.

{Received 4 January 1980, revised 10 March 1980)

cellular morphology in haploid amphibian...

Documents