Chromosome studies of the lethal hybrid Rana pipiens ♀ × Rana catesbeiana ♂

DEVELOPMENTAL

BIOLOGY

20,

Chromosome pipiens

al-517

Studies

of the Lethal Hybrid

9 X Rana catesbeiana

JAMES K. REYNHOUT’ Division

(1969)

Rana

8 ’

AND DONALD L. KIMMEL,

JR.”

of Biological and Medical Sciences, Brown University Providence. Rhode Island 02912 Accepted May 27, 1969 INTRODUCTION

Many amphibian interspecific hybrids cease normal development before or shortly after the onset of gastrulation (Moore, 1955). The failure of lethal hybrids to develop beyond gastrulation may be causally related to chromosomal breakage and loss during cleavage. Ferrier (1967) reported that urodele hybrid embryos, Pleurodeles waltlii 9 x Euproctus asper 8, become aneuploid before they are arrested at the late blastula stage. A variety of experimental situations that cause amphibian development to stop in this critical period also result in abnormal karyotypes: e.g., polyspermy (Fankhauser, 1945), interspecific nucleocytoplasmic hybrids (Hennen, 1963), nuclear-transplant embryos whose development is initiated with nuclei from more advanced cell types of the same species (Briggs et al., 1961; DiBerardino and King, 1965, 1967; Subtelny, 1965), and protein injection into zygotes (Ursprung and Markert, 1963; Kimmel, 1964a). There may be no common relationship among these systems, but it is possible that development halts subsequent to the involvement of abnormal chromosomes in nucleocytoplasmic interactions during cleavage. Amphibian interspecific hybrids that develop normally to larval stages or beyond do so without evidence of chromosomal damage. Rana pipiens 0 X Bufo americanus 13 hybrids retain complete chromosomal complements from both parental species (Kiley and ’ This investigation was supported by National Science Foundation research grant GB-3195, and by National Aeronautics and Space Administration Training Grant Ns G (T)-127. ’ Part of this research was submitted to the Graduate School, Brown University, in partial fulfillment of the requirements for the degree of Master of Science. ” Reprint requests should be addressed to Dr. Kimmel. 591

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JR.

Wohnus, 1968), and such is probably also the case for Rana pipiens 9 X Rana palustris b hybrids (Hennen, 1965). There is insufficient information, however, to permit useful conclusions about the genetic defect of amphibian hybrids that are arrested about the time of gastrulation. Hybrids between Rana pipiens 0 X Rana catesbeiana 8 display aberrant metaphase and anaphase configurations, and the accumulation of interphase chromatin at the periphery of the nuclei after their arrest as early gastrulae (King and Briggs, 1953). Kimmel (1964b) noted aneuploidy, acentric fragments, and ring chromosones in twelve reciprocal hybrids between Rana pipiens and Rana syluatica 8-10 hours before their arrest at the late blastula stage. Barbieri and Brauckman (1966), however, reported diploid chromosome numbers (2n = 22) in the hybrid between Bufo arenarum 9 x Leptodactylus chaquensis 8, which is arrested at the early gastrula stage; their methods permit no conclusions about the physical integrity of the chromosomes, and they did not confirm retention of haploid sets of chromosomes from both parental species. This paper presents karyotypes of white blood cells of the American bullfrog, Rana catesbeiana Shaw, and the leopard frog, Rana pipiens. Observations are made on the chromosomal composition of hybrids between these two species. All such hybrids are arrested at the onset of gastrulation (Rugh and Exner, 1940; Moore, 1941), yet appear to retain complete, intact haploid sets of chromosomes from both parental species. MATERIALS

AND

METHODS

Adults of Rana pipiens (J. M. Hazen, Alburg, Vermont) and Rana (Carolina Biological Supply Co., Burlington, North Carolina) were obtained in the fall and kept at 4°C in tap water and antibiotics. Ova were obtained from Rana pipiens by stripping females 24-48 hours after injecting 5.0 mg of progesterone (Sigma) in corn oil (Mazola) into the dorsal lymph sac, and a single pituitary gland into the body cavity (G. Nate, personal communication). Ova were fertilized with a sperm suspension of the appropriate species in 10% B & C solution (Brown and Caston, 1962). No fertile Rana catesbeiana ova were obtained. Preparations of chromosomes from white blood cells were made by a technique developed for mouse bone marrow by Tjio and Whang (1962). Frogs that had been kept at 4% for 6 months or more were catesbeiana

CHROMOSOMES

IN LETHAL

HYBRID

503

injected with 0.5 ml of l.Or;’ colchicine (Fisher) in amphibian Ringers (Hamburger, 1960) into the body cavity. After 24 hours at room temperature, femurs and tibiae were removed, the ends were excised, and the marrow cavity was flushed out with 2.0”; sodium citrate. The resulting cell suspension was centrifuged at approximately 800 g for 10 minutes. The supernatant was removed, and the pelleted cells were resuspended for 10 minutes in distilled water, white cells being permitted to swell and red cells to lyse. After recentrifugation, the water was decanted and about 0.5 ml of fixative (methanolacetic acid = 3 : 1) was added down the side of the tube without disturbing the loose white blood cell pellet. Thirty minutes later all but about 0.1 ml of fixative was pipetted away, and the cells were resuspended in the remainder. One or two drops of the final cell suspension were placed on slides that had been dipped into distilled water and were immediately dried by ignition. After drying in air, slides were stained 8-10 minutes in fresh Giemsa (Fisher), dehydrated through acetone and xylene, and mounted in balsam. Metaphase figures without chromosome overlap were photographed, and karyotypes were constructed from enlargements. Chromosome preparations from hybrid and Rana pipiens embryos were made by a method modified from that of Hungerford and DiBerardino (1958). Embryos that had developed for 18 hours at 18°C in 10”; B & C were treated with Cart and Mg’+-free Niu and Twitty solution (Hamburger, 1960). A concentrated suspension of animal hemisphere cells was fixed for 30 minutes in approximately 0.5 ml of 45c; glacial acetic acid, Single drops of the fixed cell suspension were covered with No. 2 square coverslips on clean slides and squashed with thumb pressure. Slides were transferred to a block of Dry Ice and, when frosted, the coverslip was removed with a cold razor blade. Slides were then air-dried, Giemsa-stained, and mounted as in the white blood cell procedure. No embryos were exposed to colchicine. Attempts were made upon embryonic tissue to promote chromosoma1 condensation and dispersal, and to eliminate the tedious squashing procedure. Treatment with coumarin (Eastman, saturated solution in B & C, Sharma and Bal, 1953), spermine tetrahydrochloride (Calbiochem, 0.5-5.0 mA4 in B & C; Davidson and Anderson, 1960), distilled water (up to 1 hour), and sodium lauryl sulfate (7 x lo-“:;, Fisher), either alone or in combination, did not improve the appear-

504

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AND

KIMMEL,

JR.

ante of metaphase figures. Incubation for 2 hours at 18’C in colcemid (Ciba, 450 rg/ml B & C) prior to squashing resulted in doubling the frequency of metaphase figures and in the appearance of the “c-metaphase” chromosomal configuration; the frequency of metaphase figures suitable for chromosomal study was decreased lo-fold over controls incubated without the drug because chromosomes were tightly clumped together. Ignition-drying or air-drying after fixation in methanol and acetic acid did not result in better spreading. Slides were scanned under bright-field illumination with a Wild M-20 compound microscope, and metaphase figures in which the chromosomes could be individually distinguished were photographed at 500 X and 1250 X using panatomic X (Kodak) and a yellow filter (Wild No. 8057). Figures from which the chromosomes were scattered, or those which lay too close to others to permit accurate assignment of their respective chromosomes, were not used. Chromosome counts were made both from enlarged photographs and at the microscope. Karyotypes were constructed from suitable photographic enlargements by arranging chromosomes in descending order on the basis of total length. Chromosomes were further enlarged by projection of the negatives upon a screen (17,000 x total enlargement; 1 inch equals 1.46 P chromosome length), and individual chromosome lengths were measured with an Addimult map mileage computer. Relative chromosome lengths were computed as the percentages of half of the total length of all chromosomes. RESULTS

White Blood Cell Karyotypes Figure 1 is a representative karyotype from white blood cells of adult Rana pipiens. Thirteen pairs of homomorphic chromosomes are present, as previously described for embryonic, larval, and adult cells of Ram pipiens (DiBerardino, 1962; DiBerardino et al., 1963; Hennen, 1964). There are no obvious changes in relative lengths of these white blood cell chromosomes, compared to those of earlier developmental stages of Rana pipiens, even though colchicine was used in obtaining the karyotype in Fig. 1. The four chromosomes placed in pairs Nos. 6 and 7 are all about the same length, but those in pair No. 6 appear to be metacentrics and those in pair No. 7 to be submetacentrics (corresponding to pairs Nos. 4 and 9 of DiBerardino, 1962). Chromosomes of pair No. 8 are less telomeric, but about the

CHROMOSOMES

IN LETHAL

HYBRID

505

FIG. 1. Metaphase from Ranu pipiens white blood cell. Arrow indicates one chromosome of pair No. 10.

same length as those of pair No. 9 (pairs Nos. 5 and 11 of DiBerardino, 1962). Pair No. 10 chromosomes contain prominent constrictions in the long arms, separated from the centromere by a short length of densely staining chromatin, as reported by DiBerardino (1962). It is difficult to distinguish among the six shortest chromosomes, those of pairs Nos. 11-13 (pairs Nos. 6, 12, and 13 of DiBerardino, 1962). The karyotype of white blood cells of adult Ram catesbeiunu also displays 13 pairs of homomorphic chromosomes (Fig. 2). Two pairs, Nos. 7 and 10, always contain prominent constrictions, resulting in satellite formation at the tips of these 4 chromosomes. Pair No. 7 is easily distinguished from No. 10, since the former are longer, submetacentric chromosomes, with constrictions in the shorter arms extending very close to the region of the centromere; constrictions in the metacentric No. 10 chromosomes are in the longer arms, and are separated from the centromere by a short Iength of densely staining chromatin. Pair No. 7 has no counterpart in Rana pipiens white blood

506

REYNHOUT

AND

KIMMEL,

JR.

cell karyotypes. The lightly staining region in acrocentric pair No. 9 extends from the proximal portion of the long arms, through the centromere, and encompasses the entire short arms of these chromosomes; this characteristic is not evident m-pair No. 9 of Rana pipiens. Constrictions and differences in reactions to stain in other chromosomes are not consistently seen. There are no other characteristics (e.g., centromere position, relative chromosome length) that distinguish between chromosomes of the karyotypes of the two species (compare Figs. 1 and 2; see Figs. 4-7). Embryo

Chromosome Number and Morphology

Twenty-six Ranu pipiens (control) and 27 hybrid late blastulae were selected for use in this study because of suitable chromosome spread and uniformity of preparative technique. Fourteen of the control embryos yielded at least one metaphase figure in which 26 chromosomes could be accurately counted; 4 others contained at least one figure in which the chromosome number was aberrant. One Ranu

6

7

8

9

10

FIG. 2. Metaphase from Ram catesbeiam

11-12-13 white blood cell

CHROMOSOMES

IN LETHAL

507

HYBRID

control, for example, was hypodiploid, and contained a ring chromosome in several metaphase figures. Nineteen of the 27 hybrid embryos yielded at least one metaphase figure in which 26 chromosomes were present; 5 had at least one figure with fewer than 26. One “hybrid” contained 13 chromosomes in all figures that could be counted; its nuclei were distinctly smaller than those of either hybrids or leopard frog controls at the same stage, and no figure was suitable for karyotyping. No metaphase figure obtained from hybrid embryos contained grossly abnormal chromosome types, such as rings or acentric fragments. Table 1 presents chromosome numbers from metaphase and anaphase figures from embryos selected for karyotype preparation and from others that provided high numbers of countable figures.

pipiens

Karotypes from Embryos

A karyotype representative of the Rana pipiens late blastula is presented in Fig. 3. Chromosomes are paired on the basis of similar lengths, and the pairs are arranged in descending order. The large constriction in pair No. 10 is striking; others were not consistently present. TABLE 1 CHROMOSOME COUNTS IN CONTROL AND HYBRID EMBRYOS Exact count Embryo

yf-

Metaphase

-___ Anaphase

figures 24 Control G-l G-9 G-12 1 3 Totals

17 14 12 6 8 57

Hybrid 5 6 8 131 127 Totals

7 10 8 17 9 51

25

26

27

52

1

3 3 5 3 2 16

1 1

1 2 1 7 4 15

1

1 1

Approximate Metaphase

Fewer

2 1 3

1 1 1 2 1 4

2

count Anaphase

25-27

50-54

11 8 3 1 4 27

3 3 1 1 2 10

4 6 6 6 3 25

1 1 1 3

508

REYNHOUT

AND

KIMMEL,

JR.

Nine hybrid karyotypes were prepared from 6 late blastulae. All contained 26 chromosomes, none of which could be classified as ring chromosomes or acentric fragments. Two of these karyotypes are reproduced in Figs. 4 and 5, with chromosomes again paired on the basis of individual length. Three chromosomes with prominent constrictions are constant features; one falls into pair No. 7 and two into pair No. 10 on the basis of length. In those karyotypes in which it was possible to determine the positions of the centromeres in the three chromosomes, the con-

FIG. 3. Metaphase

from Rana

pipiens late blastula (embryo No. 2-5)

CHROMOSOMES

IN LETHAL

HYBRID

509

striction in the shorter two of the three was separated from the centromere by a short length of densely staining chromatin (corresponding to that of chromosomes in pair No. 10 from white blood cell controls). The constriction in the third chromosome appears to include the centromere (Fig. 5), like homologs of pair No. 7 from Ram catesbeiana white blood cells (see Fig. 2). There was no homolog for this No. 7 chromosome in any of the karyotypes prepared from hybrid embryos. The large constriction in chromosomes of pair No. 10 separates each chromosome into two densely staining segments, the longer of

FIG. 4. Metaphase from hybrid late blastula (embryo No. 131)

510

REYNHOUT

FIG. 5. Metaphase

AND

KIMMEL,

JR.

from hybrid late blastula (embryo No. 157)

which contains the centromere (see Figs. l-5). The longer segments look as though they are of different lengths in karyotypes from hybrids (Figs. 4 and 5), but not in karyotypes from Rana pipiens embryos (Fig. 3). Consequently, the longer segments of the two chromosomes in pair No. 10 were measured, and the difference in their lengths determined, for each of five karyotypes from Ranu pipiens and seven from hybrid embryos. The difference in length is significant, by Student’s t test, in hybrid karyotypes (p
CHROMOSOMES

IN LETHAL

HYBRID

511

4, 6, and 12 in Figs. 4 and 5, and Nos. 2, 5, and 8 in Fig. 5 as well) displayed small constrictions or extended lightly staining regions; these were inconstant morphological features. The lightly staining region seen in chromosomes of pair No. 9 in bullfrog white blood cell karyotypes (Fig. 2) often could not be distinguished in these blastula preparations (compare homologs in pair No. 9, Fig. 5). Comparison of homolog lengths as a means of determining the species of chromosome origin is not justified, since the length of blastula chromosomes varies and since there are no control data from bullfrog blastulae. The relative lengths of chromosomes from karyotypes of hybrid embryos were compared with those of Ram pipiens to further examine the possibility of loss or extensive translocation of chromosomal

CHROMOSOME

PAIR

FIG. 6. Relative chromosome lengths from karyotypes of four &mu pipiem late blastulae. Each point is the average for both homologs. Solid line represents values published for Rana pipiem (Ursprung and Markert, 1963).

512

REYNHOUT

AND

KIMMEL,

JR.

material in the hybrids. Real lengths of the chromosomes from hybrid karyotypes fall within the range published for Ram pipiens blastula chromosomes (DiBerardino, 1962). Chromosome lengths from karyotypes of four control (Fig. 6) and four hybrid (Fig. 7) embryos are plotted together with values previously published for Rana pipiens (solid lines in Figs. 6 and 7, from Ursprung and Markert, 1963). There is little difference among the plots. Constrictions account for the relatively high position of chromosomes in pair No. 10. All the above results were obtained from embryos that had developed for 18 hours at 18°C. Hybrid embryos are arrested at about 26 hours of development at 18%. Chromosomes prepared from hybrid embryos that were 22-25 hours old were clumped together, and ac-

18 Symbol

16 -

- Embryo

.=

No.

5-5

: : ;;; 0 = 157

6-

4-

2r

00

a

1234567 CHROMOSOME

9

IO

11

12

13

PAIR

F’Ic. 7. Relative chromosome lengths from karyotypes from four hybrid late blastulae. Each point is the averaie for both homologs. Solid line representa values published for Ranu pipiew (Ursprung and Markert, 1963).

CHROMOSOMES

IN LETHAL

HYBRID

513

curate counts were usually impossible. No metaphase figure was suitable for karyotypic analysis. No deviation from the diploid chromosome number could be established, however, and no abnormal chromosome types could be distinguished in these preparations. DISCUSSION

The karyotype from white blood cells of adult Rana pipiens presented here is very similar to the ones from tadpole tail cells (DiBerardino, 1962) and adult bone marrow cells (DiBerardino et al., 1963). The karyotype from white blood cells of adult Rana catesbeiam, on the other hand, differs from the karyotype prepared from white blood cells of adult Japanese Ram catesbeiam (Seto, 1965). Chromosomes of pair No. 7 in the single karyotype Seto presented (1965, Fig. 21, p. 442) seem to show the constriction we have described, but he did not comment upon it. The large constriction in pair No. 10 is not apparent in the Japanese bullfrog karyotype. Our preparations lead us to an interpretation different from Seto’s: either Ram catesbeiam Shaw from Japan is a subspecies karyotypically distinct from the American bullfrog, or both should be included among those species with prominent constrictions in at least one chromosome pair. A constant chromosome number (2n = 26) is apparently conserved throughout the development of both species. Chromosomes from white blood cells display only the most prominent of the constrictions seen in chromosomes from blastulae. Many of the constrictions observed in chromosomes prepared from embryos could not be used to distinguish the chromosomes of one species from another because of inconstancy of appearance, chromosome overlap, and the absence of information about their presence in the bullfrog embryo. Chromosomes from late blastulae are difficult to spread for counting and karyotype preparation; positive localization of the centromere is often impossible. Nevertheless, several lines of evidence support the conclusion that the genome of this lethal hybrid is composed of undamaged haploid chromosome sets of both parental species: First, examination of anaphase and metaphase chromosomes from hybrid embryos revealed no evidence of breakage or loss. The expected diploid number was seen with a frequency greater than that of controls. No fragments, ring chromosomes, or anaphase bridges were seen. Second, individual chromosomal lengths, and their relative lengths in a karyotype, were virtually identical to those of controls.

514

REYNHOUT

AND

KIMMEL,

JR.

Third, one homolog of pair No. 7 in hybrid karyotypes contained a secondary constriction, as do chromosomes in pair No. 7 from karyotypes of bullfrog white blood cells. Even if homologs were mispaired in some hybrid karyotypes, there is no homolog with a constriction for this No. 7 chromosome: control bullfrog karyotypes have four chromosomes with prominent constrictions; control leopard frogs have two; hybrids have three. Fourth, the longer segments in the two chromosomes in pair No. 10 in hybrid karyotypes are of significantly different lengths. The position of the constriction in No. 10 chromosomes from bullfrog embryos has not been determined, but it appears that it will be different from Rana pipiens. If so, it will confirm our use of this cytological marker as evidence for retention of chromosomes from both parental species. Androgenetic haploid hybrids between these two species (Ram pipiens ( 0 ) X Rana catesbeiana a), and nucleocytoplasmic chimeras made by transplanting a diploid nucleus from a bullfrog blastula into an enucleated leopard frog ovum, are arrested at the early gastrula stage (Briggs and King, 1952). Arrested development of the Ranu pipiens 0 x Rana catesbeiana 8 hybrid occurs at about the same stage, and might follow elimination of the haploid chromosome set supplied by the ovum, with or without endomitotic replication of the remaining bullfrog haploid set. Results presented here, albeit including only small percentages of the total cell populations of hybrid embryos, make this possibility unlikely; chromosomes from both parental species remain in the hybrid several hours before arrest. These results also diminish the possibility that arrest follows loss of genetic material that is not cytologically apparent. When chromosomal aberrations accompany developmental arrest soon after cleavage, karyotypic changes usually are obvious and diverse, and are reflected in stable genetic alterations. Conversely, when normal chromosome number and integrity are maintained during cleavage of interspecific combinations, there may be no inheritable genetic change even though the embryos fail to develop normally. For example, diploid nuclei from Rana pipiens embryos may be transplanted into ova of either Rana syluatica or Ranu palustris from which the female pronucleus has been removed. Both combinations are arrested early in development. The nuclei from Rana pipiens that have cleaved in Rana sylvatica cytoplasm are both aneuploid and unable to support normal development when transplanted back

CHROMOSOMES

IN LETHAL

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515

into enucleated Rana pipiens ova (Moore, 1958; Hennen, 1963). Neither the karyotypic abnormality nor the developmental deficiency characterize the nuclei from Rana pipiens that have undergone cleavage in Rana palustris egg cytoplasm (Hennen, 1965, 1967). Similar experiments on nucleocytoplasmic combinations between Rana pipiens and Rana castesbeiana would extend our observations to the functional level. Further experimentation with amphibian nucleocytoplasmic chimeras-regardless of the method of their construction-is apt to be most fruitful if the obscure mechanism of genetic failure to support normal development is investigated at the biochemical level. Hybrids between Rana pipiens and Rana catesbeiana may be arrested without incurring either cytologic or genetic abnormality. If so, the nuclear and mitotic abnormalities seen after arrest of this hybrid combination (Briggs and King, 1952) are probably one consequence, not the cause, of earlier nucleocytoplasmic dysfunction. And, these embryos will be available for comparison of molecular events during cleavage of the hybrid and other nucleocytoplasmic combinations without the complications of damage or loss of genetic material. Those events necessary for cleavage of amphibian embryos may be distinguishable from those prerequisite for normal gastrulation and subsequent development, giving insight into the mechanisms of genetic failure to support normal early development as well as their relationships to chromosomal aberrations. SUMMARY

Developmental arrest of amphibian interspecific hybrids about the time of gastrulation may be causally related to chromosomal breakage and loss during cleavage. This paper presents the karyotypes of white blood cells of adult Ranu pipiens and Rana catesbeiana, and evidence that hybrids between Rana pipiens 0 X Ranu catesbeiana 8 retain complete haploid chromosome sets from both parental species shortly before arrest at the early gastmla stage. Both species have 13 pairs of homomorphic chromosomes. Individual real and relative chromosome lengths are very similar in both species, as are centromere positions in the two karyotypes. Two chromosome pairs in Ram catesbeiana (Nos. 7 and 10) contain prominent constrictions; the constrictions are present only in chromosomes of pair No. 10 in Rana pipiens. Metaphase figures from hybrid blastulae contained no ring chromosomes or acentric fragments; no anaphase bridges were seen.

516

REYNHOUT

AND KIMMEL,

JR.

Most figures in which chromosomes could be counted contained 26 chromosomes, and the frequency of figures with abnormal chromosome numbers was less than that from control Ranu p&ens blastulae. Relative lengths of individual chromosomes from hybrid karyotypes were the same as those from controls. Karyotypes from hybrid embryos contained three chromosomes with prominent constrictions, one falling into pair No. 7-for which there was no homolog with a constriction-and two into pair No. 10. The lengths of the longer chromosome segments of hybrid pair No. 10 were significantly different from one another; those of controls were not. The conclusion that functional inadequacy of nucleocytoplasmic interactions during cleavage can account for early developmental arrest without associated karyotypic abnormality is discussed. The relationship of chromosomal damage to blastula and gastrula arrest in other experimental systems is considered, and the Rana pipiens 0 X Rana catesbeiana b hybrid is suggested as a system amenable to further study of genetic dysfunction before gastrulation. The authors wish to express their appreciation for the advice of Dr. George Hagy, the technical assistance of Mrs. Sheryl Griffith, and the detailed critical comments of Drs. Sally Hennen and John R. Coleman. REFERENCES BARBIERI, F. D., and BRALJCKMAN,E. S. (1966). Hybridization between Bufo arenarum and Leptodactylus chuquensis. Acta Embryol. Morphol. Exptl. 9, 31-36. BRIGGS, R., and KING, T. J. (1952). Transplantation of living nuclei from blastula cells into enucleated frogs’ eggs. Proc. Natl. Acad. Sci. U.S. 38,455-463. BRIGGS, R., KING, T. J,, and DIBERARDINO, M. A. (1961). Development of nuclear

transplant embryos of known chromosome complement following parabiosis with normal embryos. In “Symposium on Germ Cells and Development” (S. Ranzi, ed.), pp. 44-447. Inst. Intern. d’Embryologie and Fond. A. Baselli, Milan, Italy. BROWN, D. D., and CASTON, J. D. (1962). Biochemistry of amphibian development. I. Ribosome and protein synthesis in early development of Rana pipiens. Develop. Biol. 5, 412-434. DAVIDSON, D., and ANDERSON, N. G. (1960). Chromosome coiling: abnormalities induced with polyamines. Exptl. Cell Res. 20, 610. DIBERARDINO, M. A. (1962). The karyotype of Ranu pipiens and investigation of its stability during embryonic differentiation. Deuelop. Biol. 5, 101-126. DIBERARDINO, M. A., and KING, T. J. (1965). Transplantation of nuclei from the frog renal adenocarcinoma. II. Chromosomal and histologic analysis of tumor nuclei in transplant embryos. Deuelop. Biol. 11, 217-242. DIBERARDINO, M. A., and KING, T. J. (1967). Development and cellular differentiation of neural nuclear-transplants of known karyotypes. Deuelop. Biol. 15, 102-128. DIBERAFCDINO,M. A., KING, T. J., and MCKINNELL, R. G. (1963). Chromosome studies of a frog renal adenocarcinoma line carried by intraocular transplantation. J. Natl. Cancer Inst. 31,769-789.

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FANKHAUSER, G. (1945). The effects of changes in chromosome number on amphibian development. Quart. Rev. Biol. 20, 20-78. FERRIER, V. (1967). l&de cytologique des premiers stades du d&eloppement de quelques hybrides lhtaux d’Amphibiens Urodeles. J. Embryol. Exptl. Morphol.

18, 221-251. HAMBURGER, V. (1960). “A Manual of Experimental

Embryology,” 2nd ed., p. 35. Univ. of Chicago Press, Chicago, Illinois. HENNER, S. (1963). Chromosomal and embryological analyses of nuclear changes occurring in embryos derived from transfers of nuclei between Ranu pipiens and Rams

syluatica. Develop. Biol. 6, 133-183. HENNEN, S. (1964). The karyotype of Rama sylvatica. J. Heredity 55, 124-128. HENNEN, S. (1965). Nucleocytoplasmic hybrids between Ranu pipiens and Rana palustris. I. Analysis of the developmental properties of the nuclei by means of nuclear transplantation. Develop. Biol. 11.243-267. HENNEN, S. (l967’). N&ear transplantation studies of nucleocytoplasmic interactions in amphibian hybrids. In “The Control of Nuclear Activity” (L. Goldstein, ed.),

pp. 353-375. Prentice-Hall, Englewood Cliffs, New Jersey. HUNGERFORD. D. A., and DIBERARDINO, M. A. (1958). Cytological effects of prefixation treatment. J. Biophys. Biochem. Cytol. 4,391-400. KILEY, S., and WOHNUS, J. F. (1968). Chromosomal analysis of Rana pipiens, Bufo americanus and their hybrid. Cytogenetics 7, 78-90. KIMMEL, D. L., JR. (1964a). Developmental arrest of protein-injected Ranu pipiens embryos. J. Exptl. Zool. 15’7,361-374. KIMMEL, D. L., JR. (1964b). Dissertation, The Johns Hopkins Univ., Baltimore, Mary-

land. KING, T. J., and BRIGGS, R. (1953). The transplantability of nuclei of arrested hybrid blastulae. J. Exptl. 2001. 123, 61-78. MOORE, J. A. (1941). Developmental rate of hybrid frogs. J. Exptl. Zool. 86, 405-422. MOORE, J. A. (1955). Abnormal combinations of nuclear and cytoplasmic systems in frogs and toads. Advan. Genet. 7, 139-182. MOORE, J. A. (1958). Transplantation of nuclei between Rana pipiens and Rana sylvatica. Exptl. Cell Res. 14, 532-540.

RUGH, R., and EXNER, F. (1940). Developmental effects resulting from exposure to X-rays. II. Development of leopard frog eggs activated by bullfrog sperm. Proc. Am. Phil. Sot. 83, 607-619. SETO, T. (1965). Cytogenetic studies in lower vertebrates, II. Karyological studies of several species of frogs. Cytologia 30, 437-446. SHARMA,A. K., and BAL, A. K. (1953). Coumarin in chromosome analysis. Stain Technol. 28, 255. SUBTELNY, S. (1965). On the nature of the restricted differentiation-promoting

ability of transplanted Rana pipiens nuclei from differentiating endoderm cells. J. Exptl. Zool. 159, 59-92. TJIO, J., and WHANG, J. (1962). Chromosome preparations of bone marrow cells without prior in oitro culture or in vivo colchicine administration. Stain Technol. 37, 17-20. URSPRLJNG, H., and MARKERT, C. L. (1963). Chromosome complements of Rana pipiens embryos developing from eggs injected with protein from adult liver cells. Develop. Biol. 8, 309-321.