INDUCTION OF MUTATIONS BY y-RAYS IN PREGONIAL GERM

Copyright 0 1983 by the Genetics Society of America

INDUCTION OF MUTATIONS BY y-RAYS IN PREGONIAL GERM CELLS OF ZEBRAFISH EMBRYOS

CHARLINE WALKER AND GEORGE STREISINGER

Institute of MoiecuIar Biology, University of Oregon, Eugene, Oregon 97403

Manuscript received May 31, 1982 Accepted September 27,1982

ABSTRACT

Specific locus (gol-1) germ line mutations are induced by y-rays with high frequencies (about IO-' r-l) during cleavage divisions in zebrafish. Mutant clone sizes range from 3 to 50% of the total number of germ cells, with a mean of about lo%, when embryos are exposed between the 16 and lo3 cell stages. About five pregonial cells are calculated to be present during the cleavage period.

N the first paper of this series (CHAKRABARTI et al. 1983) we described the I induction of mutations in mature germ cells of zebrafish, a vertebrate particularly suited for genetic analysis (STREISINGER et al. 1981). Mutants were recognized directly by virtue of the altered phenotypes of individuals that developed from mutagenized germ cells.

Here we describe mutagenesis in pregonial cells irradiated during cleavage stages followed by recovery of mutants among the progeny of adult zebrafish that developed from mutagenized embryos. We developed this alternative strategy because of the following desirable features: (1) Clones of mutants are produced, making it easier to identify and recover mutations resulting in slight alterations. (2) Deleterious or recessive lethal mutations, once identified in a clone, can be recovered from the mutagenized parent by crosses. (3) DNA heteroduplex heterozygotes are converted to homozygous duplexes and are separated from induced cytoplasmic damage by the several cycles of cell division that intervene between mutagenesis and germ cell maturation. (4) Mutagens can be administered at constant low dose rates and to uniform populations of dividing cells.

To compare mutagenesis at cleavage stages to that in eggs and sperm, we used the same approach described in the first paper (CHAKRABARTI et al. 1983) of this series: we mutagenized wild type with y-rays and detected mutations at the goJ-1 gene by crosses of mutagenized fish to goJ-Ugo1-l partners. We identified recessive lethals among progeny fish that developed from eggs made homozygous (STREISINGER et al. 1981). In the experiments described here, the y-ray-induced gol-1 mutations, once identified, were recovered as homozygous second generation progeny without the intervention of the canonical gol-1 mutation. As in the previous paper, we found a high frequency of induced gol- 1 mutations, both in absolute terms and relative to recessive lethal mutations. Genetics 103: 125-136 January, 1983.

126 C. WALKER AND G. STREISINGER

From the clone sizes of gol-2 mutants, we conclude that throughout the period of cleavage divisions (from the 16- to the 1000-cell blastula) only a few (one to ten) cells are present that contribute to the germ cell population. We define these cells as pregonial; as will be discussed, a few gonia1 (stem) cells may be chosen at a later time of embryological development from the rapidly and uniformly dividing population of cleavage cells.

MATERIALS AND METHODS

Fish strains and methods for producing homozygotes: As described in CHAKRABARTI et al. (1983) and STREISINGER et al. (1981).

Fertilization: Sperm, expelled from anesthetized males by gentle pressure, were collected in 20- pl capillary pipettes, were transferred to cold Hanks' saline and were used for fertilization within 45 min. Eggs, obtained by applying gentle pressure to anesthetized females placed into 35-mm Lux brand plastic Petri plates, were fertilized by the addition of 0.05 ml of freshly collected sperm suspended in saline together with 0.5 ml of water (STREISINGER et 01. 1981). Eggs were incubated at 28.5". Eggs that were not undergoing cleavage (and presumably were not fertilized) were discarded, and the remaining eggs were transferred to water (glass distilled) and supplemented with 1 g 1-' sea salts (Instant Ocean, Aquarium Systems). (With recent batches of Instant Ocean sea salts the concentrations described here proved toxic. At present we supplement water with 83 mg I-' sea salts.) Fish were raised to adulthood as described in STREISINGER et al. (1981).

Irradiation: Cleaving embryos were transferred to 5-ml glass vials and were exposed to y-rays from a Cesium-137 source in a model M Gammator (Radiation International). Doses actually delivered were measured at the time of irradiation by a roentgen meter (Landsverk Electrometer Company) using a 1000-r ionization chamber.

Scoring: Scoring was performed under a low power stereoscopic microscope as described in CHAKRABARTI et al. 1983.

Mutant nomenclature: The nomenclature used for Coenorhabditis mutants (HORVITZ et al. 1979) was adapted with minor modifications.

RESULTS

Production of clones: To determine mutagenic sensitivity and to measure mutant clone sizes, developing zebrafish embryos were exposed to several doses of y-rays at various times after fertilization. The fish that developed were raised to maturity and were individually crossed to gol-2/gol-Z (STREISINGER et al. 1981) fish of the opposite sex. Several percentages of the fish that developed from cleaving embryos irradiated at 66 and 180 min after fertilization gave rise to groups of golden progeny; no groups were observed among unirradiated populations (Table 1). We conclude that y-rays are efficient mutagens during early cleavage stages. The golden individuals among the progeny of a fish are usually clonal descendants of a single golden mutation, and we will refer to these groups as clones of mutants.

No golden clones were observed among the progeny of more than a hundred fish that had been irradiated as embryos at times greater than 240 min postfer- tilization. This result suggests that the mutation frequency at later times is lower than for the earlier radiation times.

Mutant clone size distribution: We examined the clone size distribution of the golden progeny described in the previous section to determine the number of pregonial cells present at the time of radiation and to identify treatment times that would serve for eventual large scale mutant isolation. To determine clone

ZEBRAFISH PREGONIAL MUTATION

TABLE 1

Induction of gol-1 clones

127

~

Fraction No. of of irradi- fish ated em- whose No. of Mutation

bryos progeny fish with Frequency of fre- Dose that are were golden induction of quency

Time after fertilization Stock (r) viable" scored clonesb clones per r per r'

66 min

180 min

Total no. 66 and 180 min

240 min

300 min

24 hr Total no. 300 min and 24 hr

Total no. unirradiated (this series)

Total no. unirradiated (historical control)

SP I1 210 440

SP I1 215 425

Starting 450

Starting 375 565

SP I1 600 Starting 870 SP 11 425

SP I1 0 Starting 0

SP 111' 0 Startingf o

0.44 48 0.08 15 0.41 161 0.23 43 0.26 15

282

0.89 40 0.69 44 0.94 99 0.25 4

8 111

0.94 20 0.92 79

99

29 66 194

1 0.99 x 10-~ 1 1.5 x 10-4 5 1.4 x 10-~ 2 1.1 x 10-~ 1 1.5 x 10-~ iod 1.3 x io-" 1.2 x 10-~

0 0 0

0 0 Od

~~~~~~ ~ ~

a Scored at 9 days. * Clones are defined as 3% or more golden progeny.

Frequency of induction of clones per r divided by the calculated number of pregonial chromo-

The probability that the fraction of fish with golden clones is the same in the irradiated and

is the mean frequency of clones per fish per r, calculated from the total number of

some sets (see Table 2 and text).

unirradiated populations is less than 0.05 (x' test with YATES' correction).

clones weighted for dose. The 95% confidence limits are 0.54 to 2.5 x

sizes, the number of golden fish and the total number of fish were counted among the 48-hr-old progeny of individual females. The fraction of golden progeny in clones ranged from 3 to 52% (Table 2). Included in the fraction counted as golden were a low frequency of mosaic individuals present in clones of golden fish, easily distinguished because of a mixture of pigmentless and pigmented cells in the (ordinarily) pigmented retina of the eye (G. STREISINGER, F. COALE, C . TAGGART and C . WALKER, unpublished results).

There are no obvious strain, sex or dose-dependent differences in the fraction of the progeny fish that are golden, although possible sex differences cannot be ruled out because of the small numbers examined.

From the fraction of the progeny that are golden, we calculated the number of pregonial chromosome sets (chromosome sets that will contribute descendants to the population of germ cells) and pregonial cells. The number of

e 1.3 x

'From CHAKRABARTI et al. 1983.

128 C. WALKER AND G . STREISINGER

TABLE 2

Clone sizes of golden progeny

No. No. of Normalized No. of of No. of golden fraction of CalcuIated

Sex of em- gol- mo- divided golden that no. of pre- Dose Stock Clone parent bryos den saic by total survived" gonia1 cells

~ ~~~

Irradiation at 66 min 210 r SP I1 w - 2

w-3

215 r SP I1 W-6 w - 7 W-8 w - 9 w-11

425 r SP I1 w-4 w-5

450 r Starting W-10

565 r Starting W-13

Irradiation at 180 min

Irradiation at 240 min

female male

male male male

female female male male male

male

251 452

1264 1206 767 297 100 280 764 663

369

7 0 0.03 183 0 0.40

92 1 0.07 617 6 0.51

53 1 0.07 26 0 0.09 16 0 0.16 43 0 0.13

387 7 0.51 34 0 0.05

187 3 0.51

0.33

0 (<O.ll)b 0.95 1.0 0.05 0.34 0.63 0.99 0.64

0.94

18 1

7 1 7 6 3 4 1

10

1 - Mean = 5.4

"Fraction of golden embryos viable at 9 days divided by the fraction of pigmented embryos viable at 9 days.

95% confidence limits.

pregonial chromosome sets is estimated as the reciprocal of the fraction of golden fish among the progeny of individual parents, and the number of pregonial cells is half that number. The arithmetic mean of the number of pregonial cells, for the population of fish exposed at 240 min or earlier, is about 5 (Table 2).

A surprising observation is that clones comprising about half the progeny are present for developmental stages ranging from 66 (8- to 16-cell blastula) to 240 min (>lo3 cell blastula). The number of pregonial cells thus appears to be small during this entire period of development.

To determine whether the fraction of golden progeny observed among mature fish at any one sampling time reflects the composition of the entire population of germ cells present, individual fish were sampled periodically. As shown in Figure 1, the fraction of golden progeny produced by any given fish did not change appreciably over a period of months. We conclude that sets of germ cells sampled at a given time do reflect the genetic composition of the entire population of germ cells,

To test whether small clones of golden fish were due to the low penetrance of the induced gol-I mutation, the golden fish of clone W-10 (Table 2), upon maturation, were crossed to gol-I/gol-l fish. Each of the 696 offspring examined was pigmentless. The induced mutation in this small clone is fully penetrant.

The presence of mosaic fish (Table 2) was puzzling. Chromosome spreads were examined for one golden fish from each of the clones W-3, W-5, W-6, W- 7 and W-10; all had a modal number of 50 chromosomes [the diploid number observed previously for zebrafish (ENDO and INGALLS 1968)l. Each of three

ZEBRAFISH PREGONIAL MUTATION 129

a C a2 Cn E a

P 400 c

a2

0 (3 c c 0) 0

20- .!A

D

w5 0

w3 +

w10 O

w 11 A

0

w9 .A

W 6 A

w7

A

, 2 4 6 8 10

Months After Initio1 Test FIGURE 1.-Clone sizes as a function of sampling time.

mosaic fish from these clones had a bimodal distribution of counts of 25 and 50 chromosomes. We discuss later possible mechanisms for the origin of these mosaics.

Characterization of gol-1 mutants: In the preceding paper (CHAKRABARTI et al. 1983) we discussed the possibility that most y-ray-induced mutations in zebrafish are long deficiencies. A prediction that follows is that most y-ray- induced mutations are lethals. Partly to test this prediction, we are character- izing the gol-2 mutants induced in the course of the experiments presented here. We designate these mutants (y)gol-2. To isolate the y-ray-induced gol-1 mutations, and to separate them from other lesions, we crossed parents that produced gol-2 clones to goJ-Z+/gol-l+ fish of the opposite sex, screened the resulting FI progeny (upon their maturation) by crosses to gol-l/gol-l, and, finally, isolated homozygous Fa progeny (by use of the ultraviolet (UV) sperm-heat shock procedure, STREISINCER et al. 1981) from those F1 who bore (y)gol-2 mutations (Table 3). We have completed this series of crosses for the four y-ray-induced mutations that arose in the large clones W-3, W-5, W-7 and W-13. The mutants from these clones were designated goJ-Z(b6), goJ-l(b7), go]-Z(b8) and gol-Z(bZ3) respectively.

As shown in Table 4, fish carrying homozygous (y)gol-2 mutations from clones W-3, W-7 and W-13, respectively, did not survive: the mutations are recessive lethals. Each mutant died at a characteristic time during development and exhibited a characteristic syndrome. Mutant b7, derived from clone W-5, is viable. Only 17% of the adult Fl fish derived from the parent of clone W-7 proved to be heterozygous for the gol-l(b8) mutation, even though about half of the fish in the original clone were golden (Table 4). The survival of b8 heterozygotes during later development was low in subsequent crosses as well; we conclude that this mutant allele is deleterious even in a single copy.

The golden mutation in clone W-9 appeared to be a dominant lethal: none of

130 C. WALKER A N D G. STREISINGER

TABLE 3

Breeding scheme for the isolation of y-ray-induced gol-1 mutations

Germ cells from fish that produced clones of (y)gol-l gol-l+

Genotypes of FI progeny formed by crosses to (y)gol-l/gol-1+ gol-i+/gol-l+

Heterozygotes identified by screening crosses to

Homozygous (F2) progeny produced from

mutants:

goJ+/gol+: ( y)gol-l/gol- 2 +

(Y)gol-2/(Y)gol-l gol-l/gol-1:

gol- 1 +/gol- 1 + (y)goJ-l/gol+ F1 fish:

TABLE 4

Viability of (y)gol-1 mutants

Among progeny of (y)gol-l/gol-l+ fish

Fraction

metes produced by Fraction mutagen- F, adults het-

ized parent erozygous for (from Table (y)gol-1 mu-

(Ykol-1 ga-

Clone 2) tation

Produced by crosses to gol-l/gol-l

Fraction (y)gol-l /gol-l

No. of No. of Among Among viable

tested fish fish tested F, adults total viable fish

Produced by LP. HS or EP"

Fraction ( y)gol- I/( y)gol- 1

No. of Among Among viable

total viable fish fish fish tested

W-3 0.40 0.42 24 0.55 0.50 258 0.52 0 54 W-5 0.52 0.43 7 0.53 0.53 182 0.49 0.48 29 W-7 0.52 0.17 48 0.51 0.24 257 0.23" 0 315

W-13 0.52 0.75 20 0.53 0.53 581 0.48 0 84 (0.08-0.30)b

(0.51-0.93)b

The homozygous (y)goi-l/(y)gol-1 fish from clone W-7 developed extremely poorly and were difficult to classify as golden. To produce less severely deficient lethals we fertilized eggs produced by (y)goJ-Z/gol-l+ fish with normal sperm from gol-l/gol-l males and then subjected these eggs to the early pressure (EP) treatment which inhibits the second meiotic division. The resultant embryos were all triploid; the golden ones have the genetic constitution (y)gol-I/(y)gol-l/gol-l and, thus, have two doses of the lethal mutation and one of a normal allele. These are the golden fish recorded here. HS = heat shock; LP = late pressure treatment.

95% confidence limits.

the golden fish present in the original W-9 clone survived past 2 weeks, neither did golden progeny produced by the parent of that clone when crossed to other stocks of gol-2 fish. The clone W-6 similarly seemed to consist of dominant lethals. Characterization of the remaining y-ray-induced gol-2 mutants is con- tinuing and will be described elsewhere, as will be genetic evidence that the gene-centromere distance is considerably decreased (as would be expected for long deficiencies) in mutants b6 and b8 but not in the viable b7.

Frequency of induced gol-1 mutations: To compare frequencies of mutations induced in blastulae with those observed in eggs and sperm, we calculated frequencies of gol-2 mutations induced per chromosome set per r. To do this we divided the frequency of induced clones (number of fish with clones divided by number of fish screened) by the y-ray dose (in r) (Table 1). The mean mutation


frequency per fish per r was divided by 10.8, the mean number of pregonial sets of chromosomes calculated to be present at the times of treatment. We estimate 1.2 X induced gol-3 mutations per chromosome set per r. This value is within a factor of four of the 4 x lop5 calculated for the frequencies induced in eggs and sperm. The estimates of pregonial cells and frequencies of clones are based on small numbers; thus, there may be no difference in the induced frequency of mutants observed here and in the preceding publication.

Frequency of induced recessive lethals: In eggs and sperm, the frequency of induced recessive lethal mutations (occurring anywhere in the genome) was surprisingly low compared with the frequency of induced mutations at the gol- 3 site (CHAKRABARTI et al. 1983). Our observations here are compatible with these low frequencies of recessive lethals. Two of the four parents that produced large clones of (y)gol-l mutants also produced clones of independently segre- gating recessive lethal mutations; the other two did not. These recessive lethals were present as heterozygotes among the F1 derived from parents of clones (Table 3); they were discovered among sets of homozygous progeny of individual FI fish. The lethal mutation from the parent of the W-7 clone is designated let(bl6); the one from clone W-3 is designated let(b25). Double mutants gol- l(b6)/gol-l(b6); Iet(b15)/let(b25) (with both mutations originally from the same treated parent) are lethals, as are the individual mutants. The mutations b15 and b16 do not appear to be linked to gol-3.

The frequency of the nonpreselected (i.e., not (y)gol-3) lethal mutations per chromosome set is approximately 0.33. We calculated this frequency using the parents of the three large clones (W-3, W-5 and W-7) obtained upon irradiation times of 180 min or earlier. The large clone W-13 (obtained upon irradiation at 240 min) was omitted because radiation sensitivity to mutagenesis decreases at later times. Each of the parents of these three clones has two pregonial sets of chromosomes (Table 2), giving a total of six chromosome sets from which two different lethal mutations have been recovered. Dividing the fraction R by the dose gives a frequency of 1.2 x to 4.4 X measured by CHAKRABARTI et al. (1983) in eggs. We emphasize that the numbers on which the present estimate of mutation frequency is based are very small. Nevertheless, these results to demonstrate the induction of recessive lethals at sites other than gol- 1. We have observed other clones of (non gol-3) lethal mutants among homozygous progeny derived from parents that had been irradiated at 180 min of development.

We find that the survival of homozygous progeny of irradiated parents is not much lower than that of homozygous progeny of unirradiated parents. These observations exclude the possibility of much higher frequencies of lethal mutations than estimated here. More extensive experiments, which will provide more precise frequencies of induced recessive lethals, are now underway.

Spontaneous mutations: Approximately 5% of fish tested for the gol-3 mutation produced one or two golden individuals among their otherwise pigmented progeny (Table 5). The fraction of parents that produced occasional golden fish was similar for irradiated and unirradiated populations and was independent

per r (95% confidence limits = 1.5 x This frequency is similar to the 4 x

132 C. WALKER AND G. STREISlNGER

TABLE 5

Spontaneously arising golden fish

No. of par- No. of par-

Time after fertil- Dose eny fish exam- No. of Fraction progeny were duced 6 2 % No. of prog- ents whose ents that pro-

ization ( r ) ined at 48 hr golden golden examined golden progeny

0 22,268 6 2.7 X 99 5 66 and 180 min 210-450 60,385 13 2.2 x 10-~ 282 8 240 min to 24 hr 375-870 38,151 8 2.1 X 195 6

a Clones among the progeny of fish that had been irradiated are defined as induced if the clone sizes are t3% (see text).

of the time and dose of radiation. Parents that produced individual golden progeny did not produce further golden fish with a frequency higher than the population as a whole.

Karyotypes of eight individual golden progeny were examined: one had a modal number of 50 chromosomes, five had 25, one had 28 and one had 49. Only the individual with 50 chromosomes appeared viable before it was fixed for karyotyping. We conclude that most of these individuals arose as a result of failure in disjunction.

Among approximately 2.2 x lo4 control progeny examined, only one normal- appearing golden fish was observed. This fish was not sacrificed for karyotyping and survived. Assuming that viable golden fish have not experienced nondis- juction, we calculate a spontaneous mutant frequency of about 5 X on the basis of this single individual. Considering the irradiated and unirradiated populations together, of 1.2 x IO5 progeny fish screened, four singly occurring viable golden fish were observed. On the basis of this slightly larger sample, we calculate a frequency of about 3 X (95% confidence limits = 0.8 to 8.5 x

Induction of maleness: An unexpected observation was the dose-dependent induction of males by y-irradiation during cleavage stages. The selected population I1 (SPII) fish that were irradiated at the blastula stage were produced by crossing the homozygous clones C20 and C26. Ordinarily, about 99% of these SPII fish develop into females. As shown in Table 6, higher doses of irradiation result in a majority of males. The induction of maleness seems to follow higher than first order kinetics. Similar inductions of maleness were observed upon the irradiation of cleavage stage homozygous eggs derived from members of homozygous clones. The mechanisms of the genetic control of sex in zebrafish are unknown, and we will discuss elsewhere the many possible mechanisms involved in the induction of the sex change we observe.

spontaneous mutants.

DISCUSSION

lnduction of clones of mutants: The experiments described here were designed to identify developmental stages suitable for the induction of clones of mutants. We have found that mutagenesis during cleavage division stages is ideal for our purposes: mutant clone sizes range from 50 to 5% of the progeny. Mutagenesis


TABLE 6

Effect of y-rays on sex ratio

Time after fertilization Dose (r) No. examined Z Male

66 min

180 min

300 min

24 hr

0 125 210 440 95 215 425 155 295 600 205 425

141 189 143 24 222 226 72 226 219 213 198 199

1 20 42 75 19 44 95 10 19 53 33 60

at these developmental times has enabled us to isolate y-ray-induced mutations at the gol-1 gene and recessive lethal mutations at other sites. As will be described elsewhere, we have successfully mutagenized at cleavage stages with the alkylating agent ethylnitrosourea (ENU), and J. GOLIN and D. GRUNWALD in our laboratory have isolated ENU-induced mutants that were first identified as clones.

From the clone sizes of gol-1 mutants described here, we conclude that one to ten pregonial cells are present up to 4 hours after fertilization, when the total number of cells in the blastula is greater than 1000. This small number is in contrast to the much larger mean of 35 to 70 cells that contribute descendants to the pigmented epithelium of the retina of the eye ( G . STREISINGER, F. COALE, C. TAGGART and C. WALKER, unpublished results).

Our estimates of the number of pregonial cells could be biased in two ways. Radiation-induced deleterious mutations could result in a decreased rate of cell division as has been observed in Drosophila (ABRAHAMSON et al. 1966). Slowed division rates of cells that have experienced a gol-1 mutation could result in decreased clone sizes and lead to an overestimation of the number of pregonial cells. We did not observe striking correlations between clone size and deleteri- ousness, but some small clone sizes (for instance, in W-2) could be due to this cause. Conversely, if deleterious mutations that slowed division rates were induced in cells that did not experience i t gol-1 mutation, the gol-1 clone sizes would be large, leading to an underestimation of the number of pregonial cells. The observation that clone sizes remained constant in given adult individuals over periods of many months (Figure 1) argues against the intervention of strong selective effects against pregonial cells during development in most of the clones.

For the period between the 16-cell stagc and 180 min, when the total number of cells in the blastula was greater than 1000, the mean number of pregonial cells was calculated to be about 5, and a large clone was produced by a fish irradiated as late as 240 min. The limited number of cells that contribute

134 C. WALKER AND G. STREISINGER

descendants to the germ line could be due to either of two mechanisms. (1) Germ line stem cells could be chosen early (during the first few division cycles) and then remain undivided until some time later than 240 min (our last measured point). (2) Germ line stem cells could be chosen at a time, after 240 min, from the population of cells that previous to that time had been dividing rapidly. We believe that the second alternative is more probable. Cell divisions occur rapidly and synchronously at least up to the 16-cell stage, precluding the possibility of a small population of nondividing cells. Synchronous divisions are more difficult to observe at later times, but no population of singularly large cells is evident at 180 min. We believe it is probable that a small set of cells is chosen for the germ line stem cell (spermatogonial and oogonial) populations at some later time.

It is noteworthy that a mean of about five pregonial cells has been calculated for the mouse (RUSSELL 1964; SEARLE 1978). Similarly, in Drosophila (SONNEN- BLICK 1950) a small number of nuclei are chosen to become germ cell progenitors in the approximately 500 nucleus syncytium, and in Xenopus (IKENISHI and KOTANI 1979) a small number of primary germ cells can be identified at gastrulation.

The number of pregonial cells identified by clonal analysis may not equal the number of cells that enter the germ line. If cell mixing fails to occur during development, groups of contiguous cells selected for the germ line may be descended from a mutant ancestor by chance, leading to an underestimation of the number of gonia1 cells present (LEE 1976; RUSSELL 1964). In zebrafish a great deal of mixing seems to occur during development, as judged by the analysis of clone size distributions in the pigmented retina of the eye (G. STREISINGER, F. COALE, C. TAGGART and C. WALKER, unpublished results).

Mosaics: Some males, irradiated as early embryos and crossed to gol-Z/gol-1 females, produced not only golden clones but also a few percent of individuals that were mosaics both for golden vs. standard pigment phenotypes and haploid vs. diploid chromosome sets. A possible mechanism of origin is that the sperm pronucleus (with a gol+ chromosome) did not migrate and fuse with the egg pronucleus but remained with its chromosome set undivided until after the first cleavage division of the maternal cell. If the sperm pronucleus then fused with one of the two maternal nuclei, a diploid gol-Z/gol+ and a haploid gol-1 cell would be formed. The defective behavior of the sperm would have to be due to the presence of the gol-1 mutation in spermatogonia, because groups of mosaic individuals were not seen in progeny that did not contain golden clones.

Mutation frequencies: In the first paper of this series (CHAKRABARTI et al. 1983) we described the frequency of y-ray-induced specific site and recessive lethal mutations in eggs. Here we show that y-ray-induced mutations occur at similar frequencies during early cleavage divisions.

In this paper we have demonstrated that the y-ray-induced events are germ line mutations by recovering the mutants among second generation progeny of mutagenized parents. The induced specifc locus mutation frequencies in zebrafish eggs, sperm and early cleavage divisions were about a 100-fold higher than has been observed for mouse oocytes or spermatogonia (RUSSELL 1965; RUSSELL 1977). The high frequencies we observe may be due to the speed with


which the early cleavage divisions occur in zebrafish: there may be less opportunity for repair during the approximately 15-min intervals between divisions than during the much longer division times in the mouse. In support of this possibility is the observation that mutation frequencies seem to be lower at later developmental stages, when cleavage divisions are less rapid.

The experiments described here confirm the unexpectedly high ratio (1:lOO) of specific site mutation to recessive lethals. We have examined only a very small number of fish, but our observations do set upper limits to the frequency of recessive lethals: a high frequency of recessive lethals would have been detected by virtue of the inviability of the homozygous F2 progeny obtained from mutagenized fish.

Nature of mutations: We have found three recessive lethals, two dominant lethals and one viable mutant among the six (y)gol-l mutations examined thus far. Analyses to be described elsewhere suggest that at least two of three additional (y)gol-1 mutations are recessive lethals, The fraction of y-ray-induced specific site mutations that are lethal in zebrafish falls within the range observed in mice for radiation-induced mutations: at the s locus in the mouse all of 38 mutations were lethal (RUSSELL and RUSSELL 1959), at d 92% (RUSSELL 1971) at c 40% (RUSSELL et al. 1979), and at se 56% were lethal (RUSSELL 1971).

The high proportion of lethals among y-ray-induced specific site mutations is compatible with the suggestion (CHAKRABARTI et al. 1983) that most y-ray- induced mutations in zebrafish are long deficiencies. Attempts to detect dele- tions cytologically are now under way.

Regardless of the nature of y-ray-induced mutations in zebrafish, their high frequency suggests that it will be feasible to isolate mutations in many genes of interest.

We are very grateful to M. CHALPIE, S. GLUECKSOHN-WAELSCH, J. GOLIN, D. J. GRUNWALD, M. F. LYON, L. NAWROCKI, E. NOVITSKI, A. RUDER, L. B. RUSSELL, K. SANKARANARAYANAN, F. SINGER and 1. G. STANLEY for suggestions concerning the interpretation of our data, references to the work of others and criticisms of this manuscript. We thank JULIE DUNN, NANCY CARETTO and ELIZABETH COOKSEY who did the typing. This work was supported by National Institutes of Health grant GM 22731.

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Corresponding editor: D. BENNETT

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INDUCTION OF MUTATIONS BY y-RAYS IN PREGONIAL GERM