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Genetic control of somatic cell differentiation in Volvox
DEVELOPMENTAL
‘72, 226-235
BIOLOGY
(1979)
Genetic Control of Somatic Cell Differentiation Analysis
of Somatic
Regenerator
ROBERT J. HUSKEYANDBARBARA Department
of Biology,
Received
January
University
of Virginia,
25, 1979; accepted
in Volvox
Mutants E. GRIFFIN'
Charlottesville,
in revised
form
Virginia March
22901
30, 1979
The somatic regenerator (reg) mutants of Volvox carteri affect the ability of the normally terminally differentiated somatic cells to establish and/or maintain the differentiated state. Thirtynine reg mutants of four phenotypic classes have been mapped to two, unlinked genes, regA and regB. Mutants at the regA locus have one of three phenotypes: All somatic cells regenerate new spheroids, somatic cells in the spheroid posterior region regenerate while those in the anterior region differentiate as somatic cells, or regenerating and nonregenerating cells are randomly intermixed. The regB mutant has a random intermixture of regenerating and nonregenerating cells. Somatic cells regenerate new Volvon spheroids in two ways; the cells lose their characteristic shape, become immotile, enlarge and undergo cleavage similar to that of normal reproductive cells or undergo cell division without prior enlargement or loss of cell shape. Temperature shift experiments on a cold-sensitive reg mutant suggest that the gene product acts after the somatic cell initials are formed at the end of cleavage.
timing of the action of the gene product or products. We report here a genetic analysis of a number of somatic regenerator mutants along with evidence as to the time of action of the gene product.
INTRODUCTION
The somatic cells of the multicellular green alga, Voluox, have the characteristics of terminally differentiated cells. These cells have a characteristic shape, specialized organelles, and a characteristic pattern of proteins, and are not capable of cell division (Starr, 1969, 1970; D. Kirk and M. Kirk, personal communication). Mutants of VolVOX carteri have been reported in which the somatic cells are capable of reversing the differentiated state in that they lose the somatic cell characteristics, enlarge, and cleave to form new spheroids (Starr, 1970; Sessoms and Huskey, 1973; Griffin and Huskey, 1974; Huskey et al., 1979; Kurn et al., 1978). These somatic regenerator (reg) mutants appear to define a developmentally important gene or genes which regulate the establishment and/or maintenance of the differentiated state. Because of the significance of this developmental function, it is important to determine the number of genetic loci involved and the
MATERIALS
226 Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
METHODS
Culture conditions. The culture conditions, mutagenesis procedures, and crossing techniques are reported elsewhere (Huskey et al., 1979). All strains are derived from Volvox carteri f. nagariensis HKlO (female) and 69-lb (male), which were generously provided by R. C. Starr from the Algal Culture Collection at the University of Texas. Temperature shift experiments were performed with spot plate cultures using illuminated incubators (Precision Scientific) providing illumination from single, 15-W fluorescent strip lamps at a maximum of 1200 candles as measured by a Goosen Pan-Lux light meter; temperature was regulated at +l”C. Mutant isolation. The initial isolation of somatic regenerator (reg) mutants was by visual examination of a nitrosoguanidine-
I Deceased.
0012-1606/79/100226-10$02.00/O
AND
HUSKEY
AND
GRIFFIN
Volvox
mutagenized culture (Huskey et al., 1979). Spheroids with regenerating somatic cells are highly visible due to their dark green appearance which results from the enlarged cells on the surface of the spheroid. This technique has the disadvantage that recently released reg spheroids are difficult to distinguish from wild type so some mutants may be overlooked. Also, there is no enrichment or selection for the reg phenotype, which means that a great number of spheroids must be examined in order to find the mutants. A slight enrichment for reg and other developmental mutants can be effected by subjecting the mutagenized culture to phototactic selection prior to isolation of mutants. The reg spheroids are not capable of motion after the somatic cells begin regeneration so they do not respond to a phototactic stimulus. However, a number of morphogenetic mutants are also nonphototactic for a number of reasons; thus enrichment for reg mutants using photoactic selection is incomplete. It is possible to achieve a high selection efficiency for reg mutants using growth pattern on agar medium as the selective criterion. One plating method is to spread a mutagenized culture on the surface of agar medium. If the moisture content of the culture dish is high enough to avoid dehydration of the spheroids, clones of Volvox will arise on the agar. reg mutants give rise to dark green clones which can be seen on the uniform green background of the other clones. Another plating method is to spread the mutagenized culture on the surface of an agar plate using the soft-agar technique; reg clones are more compact than wild-type clones and can be removed from the plate for verification of phenotype (Kurn et al., 1978). Both of the plating techniques can be used in conjunction with a regimen to isolate temperature-conditional mutants by incubating the plates at high or low temperature. RESULTS
Phenotypes
of the reg mutants.
The
Somatic
Regenerator
Mutants
227
young spheroids of all the reg mutants are nearly indistinguishable from wild type following inversion and release. However, as the reg spheroids begin to enlarge, some or all of the somatic cells begin to enlarge and lose their flagella, their eyespots usually disappear, and the production of extracellular matrix appears to slow. The number and location of the regenerating somatic cells constitute the basis for categorization of the reg mutants into four phenotypic classes. The class I mutants (33 isolates) show regeneration of all the somatic cells of the spheroids (Fig. 1). The regenerating cells enlarge and become vacuolated in a manner resembling developing gonidia. The regenerating cells rarely attain the size of a normal gonidium but they do go through a condensation of cytoplasmic material just prior to cleavage as do gonidia (Starr, 1969). The cleavage divisions of a regenerating cell are fewer in number than for a gonidium and the resulting spheroid has fewer reproductive and somatic cells than does a spheroid resulting from cleavage of a gonidium (Fig. 2). This class of mutant has been isolated by others, and two of these isolates are included in this study (Table 2). The class II reg mutants (3 isolates), which are similar to the one reported by Starr (1970)) have a polarization of regenerating cells such that the cells in the anterior portion and sometimes the extreme posterior region of the spheroid remain in the differentiated state while those cells in the posterior region undergo regeneration (Fig. 3). The somatic cells which do not regenerate occupy about one-quarter or less of the spheroid. Most of the regenerating cells of mutants in this class follow the pattern described for the class I mutants. However, some of the cells which occur at the junction of regenerating and nomegenerating cells undergo a different form of new spheroid formation. Instead of losing flagella and eyespots and enlarging to form a small, pseudogonidium, the cells begin to divide while retaining the characteristic
5
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FIG. 1. regA156 showing the class I phenotype. Some of the regenerating cells have cleaved and inverted. Daughter spheroids from the cleavage of gonidia can be seen in the interior of the parent spheroid. (The bars in Figs. 1, 3, and 5 represent LOO pm.1 FIG. 2. A spheroid resulting from cleavage and inversion of a regenerating somatic cell. Note the reduced number of gonidia (large cells in the interior) and somatic cells. (The bars in Figs. 2, 4, and 6 represent 10 pm.) FIG. 3. regA145 showing the class II or polar phenotype. The clustered, enlarged cells on the surface of the spheroid are beginning regeneration. The gonidia can be seen as large, darker cells in the interior of the spheroid. FIG. 4. Surface view of a regA of class II phenotype. Nonregenerating somatic cells (SC) are present. Some cells have enlarged and undergone cleavage (cl) to form small embryos (indicated by arrow) or inverted to form small spheroids complete with gonidial initials (to right of indicated embryo). Some cells regenerate by direct division (d) to form a spheroid containing only somatic cells (see Fig. 6). Daughter spheroids resulting from normal gonidial development can be seen underneath the surface cell layer. FIG. 5. regA141 showing the class III or random phenotype with an intermixture of somatic cells and enlarged, regenerating cells. FIG. 6. A spheroid resulting from division of a regenerating somatic cell without prior enlargement (compare to d of Fig. 4). Gonidia are not present. 228
HUSKEY
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Volvor
shape of somatic cells (Fig. 4). Each daughter cell has two flagella, but only one of the cells of the growing individual contains an eyespot. When division ceases, the resultant spheroid contains 20-50 cells, but few if any obvious reporductive cells are present in the interior of the spheroid (Fig. 6). These apparently sterile spheroids, which resemble the palmella stage of Chlamydomonas or the adult forms of other colonial members of the Volvo&es (PickettHeaps, 1975), usually develop reproductive cells resembling gonidia or regenerating somatic cells. Mutants of the class III type (2 isolates) have a near-equal number of regenerating and nonregenerating cells, with a random, intermixed distribution of the two cell types (Fig. 5). The cells regenerate in the patterns of the class II type. The class IV reg mutant resembles the class III mutants with an intermixture of regenerating and nonregenerating cells, but regeneration occurs much earlier in the life cycle and the nonregenerating cells appear not to fully differentiate. For example, the spheroids are motile for a very short time and very little spheroid expansion is evident, suggesting that there is little production of extracellular matrix. The class IV mutant is very sensitive to agitation and breaks up into single cells in an aerated culture. Genetic analysis of reg mutants. The only diploid phase of the Volvox life cycle is the zygote which germinates by meiosis to yield one haploid zoospore (Starr, 1975). Therefore, strains carrying single site mutations should show a 1:l segregation of progeny when crossed to wild type; crosses of allelic mutants will give all mutant progeny unless intragenic recombination occurs; crosses of unlinked mutants of similar phenotype should result in a 3: 1 ratio of mutant to wild-type progeny; and crosses of mutants in linked genes will result in a mutant: wild-type ratio between the two extremes (Huskey et al., 1979). All of the reg mutants behave as single
Somatic
Regenerator
229
Mutants
gene mutants in crosses with wild-type, showing a 1:l segregation of reg to wild type (Table 1). A similar finding was reported by Starr (1970) with a reg mutant which is included in this study, regA154. These results mean that none of the reg mutants reported here are due to multiple TABLE ONE-FACTOR reg parent
regAlO1 regA102 regA 103 regA104 regA 105 regAlO8 regAlO7 regA 108 regA 109 regAll1 regAll2 regAll3 regAl14 regA115 regAl16 regAl17 regAll8 regAll9 regA123 regA125 regA126 regA128 regA135 regA137 regA138 regAl41 regA142 regA145 regA147 regA149 regA150 regA 153 regA154 regA155 regA156 regA157 regA159 regAl60 regB144
Number
1 CROSSES” ofprogeny
p (>)
reg
Wild
Total
73 64 64 54 179 63 62 47 64 58 96 46 55 79 60 57 92 65 84 85 148 138 52 74 45 40 58 54 284 133 67 101 84 122 67 87 15 52 328
57 59 54 54 184 57 61 52 71 49 106 50 45 68 56 54 96 58 77 90 151 109 51 62 44 48 59 53 277 154 50 95 80 99 51 65 18 57 294
130 123 118 108 363 120 123 99 135 107 202 96 109 147 116 111 188 123 161 175 299 247 103 136 89 88 117 107 561 287 117 196 164 221 118 152 33 109 622
0.10 0.60 0.30 0.90 0.70 0.50 0.90 0.60 0.50 0.30 0.40 0.60 0.30 0.30 0.70 0.70 0.70 0.50 0.50 0.70 0.80 0.05 0.90 0.30 0.90 0.30 0.90 0.90 0.90 0.20 0.10 0.60 0.70 0.10 0.10 0.05 0.60 0.60 0.10
n In each cross the reg mutant was the female strain and was crossed to a wild-type male strain. The deviation of the progeny classes from the expected 1:l ratio was compared to the chi-squared distribution.
230
DEVELOPMENTAL
mutations genes. Pairwise performed which had type male the reg by
in unlinked
or loosely
BIOLOGY
linked
crosses of the reg mutants were using six separate reg mutants been recovered in the mating(mtm) strain (Table 2). In 57 of reg crosses, the progeny were all TABLE TWO-FACTOR
mtf
Cross
VOLUME
mutant with the exception of one wild-type recombinant. These results suggest that this set of mutants is allelic at the same genetic locus, which has been designated regA. The regA group includes all the mutants of the class I, II, and III phenotypes (Table 2). 2
CROSSES OF
1
72, 1979
reg MUTANTS”
Cross
2
Cross
3
Parent
mtm
mtm
Progeny
Parent
-
reg
+
mtm
Progeny
Parent
Progeny
Parent
-
reg
+
reg
+
regAlO1 regAll2 134 0 regA102 regAll2 0 regAlO5” 286 32 0 regAl41 b 46 1 regA 103 regAll2 407 0 regAlO4 regAlO1 83 0 regAll2 755 0 regAl41’ 85 0 regAlO5” 0 regAlO1 37 regAll1 23 0 regAl41 b 35 0 regAlO6 regAll2 403 0 regA107 regAlO1 0 regAl41 b 48 44 0 regAlO8 regAlO1 34 0 regAll2 97 0 regAlO9 regAlO1 7 0 regAll2 586 0 regAl41’ 24 0 regAll1 regAlO1 56 0 regAlO5” 9 0 regAl12 regAl42” 80 0 regAl41 b 69 0 regAll3 0 regAl41 b regAlI 345 18 0 regAll4 regAll2 0 173 regAll5 regAlO1 239 0 regAll2 242 0 regAl41’ 153 0 regAll6 0 regAl41’ regAll2 533 6 0 regAl17 regAlO1 153 0 regAll2 191 0 regAll8 regAll2 73 0 regAll9 regAlO1 80 0 regAll2 123 0 regAl23’ 0 regAll2 88 regA125 regAll2 322 0 0 regA126 regAl45” 138 regA128 regAll2 539 0 regAl41 b 31 0 regA135 regAll2 213 0 regAl41 b 205 0 regA137 regAll2 4 0 regAl41 b 75 0 regA138 regAll2 81 0 regA142” regAl41 b 148 0 regA147 regAll2 320 0 regA149 regAlO1 83 0 regA150 regAl45” 71 0 regAl41 b 0 regA153 67 regA154d regAl41’ 273 0 regAl55’ regA 149 422 0 regA156, regAll2 185 0 regA157 regAl41’ 0 154 regA159 regAll2 0 85 regAlGO regA145 171 0 regB144’ regAll2 61 24 o All regA mutants are of the class I phenotype, with the following exceptions: “regAlO5, regA123, and regA145 are mutants of class II phenotype; hregA141 and regA142 are mutants of class III phenotype; ‘regBl44 is the only class IV mutant; ‘isolated as a spontaneous mutant by R. C. Starr; ‘isolated induced mutant by D. L. Kirk; ‘isolated as a spontaneous mutant by P. O’Farrell.
as a nitrosoguanidine-
HUSKEY
AND
Volvor Somatic Regenerator Mutants
GRIFFIN
type. When the mutant is grown at the permissive temperature (37°C) and shifted to the restrictive temperature (24”C), the resulting spheroids will be mutant in phenotype if the shift occurs before the time period in which the gene product acts (the sensitive period) or they will be wild-type in phenotype if the shift occurs after the sensitive period (Suzuki, 1970). Alternatively, when the mutant is grown at the restrictive temperature and shifted to the permissive temperature, the resulting spheroids will have a wild-type phenotype if the shift is made before the sensitive period or a mutant phenotype if the shift is made after the sensitive period. The temperature shift experiments on regAlSO were complicated by two factors. First, the incubation conditions affect the generation time of wild-type as well as reg mutants. For regA150, the generation time is 48 hr when grown at 37°C or 196 hr when grown at 22°C. This fourfold elongation of the life cycle is nearly uniform for all parts of the cycle and may result from a combination of lowered temperature and slightly lowered light intensity due to differences in the illuminated incubators used. For this reason, the data presented in Fig. 7 are
The reg mutant of class IV phenotype gave a 3:l segregation of reg to wild-type when crossed with one of the regA mutants, which means that this mutant defines a second, unlinked locus, regB. Previously reported data indicate that the regA locus is not linked to the mating type locus (mt) or any other of the existing mutants of Volvox carteri (Huskey et al., 1979). Based on these results, the regA locus has been assigned to linkage group II of Voluox. The regB mutant is also unlinked to mt and segregates independently with markers from linkage groups II, III, V, VII, and VIII (data not presented). Temperature shift experiments. One of the reg mutants, regAlSO, is a cold-sensitive mutant which has the mutant phenotype when grown at 24°C but has a wildtype phenotype when grown at 37°C. This mutant strain was used in temperature shift experiments in an attempt to determine the time of action of the gene product. The basic experiment is to grow the mutant synchronously at one temperature and shift to the other temperature condition at various times after the initiation of gonidial cleavage. The resultant spheroids are scored for mutant versus wild-type phenoTIMING
OF THE
LIFE CYCLE
(hrs 1
6
I2
I6
24
30
24
46
72
96
120
OF THE LIFE
CYCLE
100
shift down (33OC) 42 46 rhlft “P (24-Z) 144 I66 I96 4 (shift up. Eg:(I)
36
(shift ,750 FRACTION
231
down,w_t) ,675
FIG. 7. Temperature shift experiments on regAl5f.T”. Gonidia from regAl5F were isolated and cultured in illuminated incubators at either 24°C (A) or 33°C (0). At the times indicated, samples were shifted to the other incubation temperature and the resulting spheroids were scored for somatic cell regeneration. A spheroid was scored as a regenerator if a sector of regenerating cells was present. The generation times for Volvox grown at the two temperatures are given at the top of the figure. The data are plotted as the fraction of the life cycle for the temperature of the original culture.
232
DEVELOPMENTAL BIOLOGY
plotted as the fraction of the life cycle rather than in hours and are based on the initial temperature of incubation. Furthermore, the beginning of the experiment is based on the initiation of cleavage of gonidia since this is a portion of the life cycle which is reproducibly identifiable in both reg and wild-type spheroids. The second problem in analyzing the temperature shift experiments is that the number of regenerating somatic cells in any one spheroid varied as a function of the length of time at the restrictive temperature. Thus, spheroids shifted to the restrictive temperature at early times prior to the sensitive period had more regenerating cells than those shifted just before the sensitive period. The reciprocal result occurred with the shifts to the permissive temperature. Because it was difficult to quantify the amount of regeneration in any one spheroid, the spheroids were scored as reg in phenotype if they had a sector of regenerating somatic cells. It should be noted here that the regAlSO spheroids show three classesof regenerator phenotype depending on the time of the shift to the restrictive temperature. Spheroids which result from early shifts to the restrictive temperature have the class II or polarized phenotype, while spheroids which result from late shifts prior to the sensitive period have the class III or random phenotype. Spheroids which result from continued growth at the restrictive temperature have the class I or complete phenotype. Thus, the one mutant strain, regAlSO, mimics all three classesof regenerator mutants mapping to the regA locus depending on the length of time at the restrictive temperature. This supports the results of the genetic analysis that the regA locus is a single gene. The results of the temperature shift experiments (Fig. 7) indicate that the sensitive period for the action of the reg gene product begins following the end of cleavage and inversion (one-fourth of the life cycle) and ends following release of the young spheroids (one-half of the life cycle).
VOLUME 72, 1979
It is during this time period that the cells develop eyespots, the characteristic cell shape becomes apparent, the cells begin synthesis of the extracellular matrix, and the chloroplast becomes morphologically distinct (Kochert and Olson, 1970). Thus, the regA gene product appears to function at the time when somatic cells begin to display a number of the characteristics of the differentiated state. An important result of the temperature shift experiments is that a regA150 embryo or spheroid which is shifted after the sensitive period does not show regeneration of the somatic cells; that is, there is a definite sensitive period. Only the somatic cells of the spheroids of the next generation show regeneration and only if maintained at the restrictive temperature. This result suggests that the reg gene product is required for the initial establishment of a stably differentiated state but is not required continuously for the maintenance of that state. When the temperature shift experiments with regA150 were performed elsewhere under conditions resembling those reported here, similar results were obtained. However, it was also observed that if incubation at both permissive and restrictive temperatures was carried out at the same high (about 1850-ft-candle) level of illumination, the frequency of expression of the regenerator phenotype was reduced (D. L. Kirk, personal communication). The significance of this result is being explored further, but it appears to indicate that the light level in some way modulates the expression of the temperature-conditional phenotype in the regAlSO mutant. DISCUSSION
The 39 independently isolated reg mutants reported here define only two, unlinked loci of the Volvox genome-regA and regB. This fact reinforces the original suggestion that the reg mutants identified a genetic function which is critical for differentiation (Starr, 1970). This is in contrast to findings in some organisms such as Neu-
HUSKEY
AND
GRIFFIN
Volvox Somatic Regenerator Mutants
rospora where mutants of similar morphological phenotype have been found to affect the enzymes of basic biochemical pathways and occur in a number of different structural genes rather than in a few developmentally important regulatory genes (Scott et al., 1973). The limited number of reg loci also lends more credence to the basis for the spontaneous mutation rate method used by Kurn et al. (1978) to suggest that the reg gene functions in the young embryo rather than in the somatic cell. The occurrence of three phenotypic variants among mutants mapping at the regA locus is suggestive of a set of closely linked genes rather than a single gene. However, two results offer evidence that the regA locus is a single gene. First, in 57 pairwise crosses of regA mutants with each other, only one wild-type recombinant was recovered in over 9000 total progeny. In the closely related organism, Chlamydomonas, intragenic recombination occurs at a level of 1% or less (Matagne, 1978). Since recombination at the regA locus is less than l%, it can be tentatively identified as a single gene using a recombinational definition. Second, the temperature-conditional mutant, which is probably due to a missense mutation, mimics the three phenotypic classes of regA mutants depending on the length of developmental time at the restrictive temperature. It is easiest to explain this behavior if only one gene is involved. Furthermore, the fact that a temperature-conditional mutant was isolated means that a gene product is made and it is likely that the ultimate gene product is a protein. The temperature shift experiments on the cold-sensitive mutant establish the time period in which the gene product acts. This period begins after cessation of cleavage, meaning that each somatic cell is probably acting independently in establishing the stably differentiated state. This also means that the unequal division which normally sets somatic and gonidial initials apart during cleavage (Starr, 1969) is not the only point at which control is exerted over the
233
reproductive potential of somatic cells. In addition, the reg gene product is not required continuously to maintain the differentiated state since the shift experiments show an end point to the sensitive period. Thus, the regA gene product acts in the somatic cell initials to establish the stable, differentiated state and, apparently, is not required for maintenance of differentiation. The suggestion that the somatic cells of reg mutants undergo dedifferentiation rather than failing to establish the differentiated state is based on the observation that somatic cells of reg mutants have the general morphology of fully differentiated somatic cells and are therefore fully differentiated. In fact, preliminary biochemical studies favor this notion of full differentiation in regA somatic cells in that these cells produce the same malate dehydrogenase isozyme seen only in wild-type somatic vegetative cells and distinct from the isozyme of gonidia (K. V. Chace, personal communication). However, the finding that some regenerating cells divide without prior enlargement and loss of characteristic shape means that some caution must be used in defining the differentiated state of Volvox vegetative cells. In addition, this finding is very significant in opening up the study of the evolution of the Volvocales. This latter point will be explored in more detail in a subsequent publication. The regenerating somatic cells of regA mutants are not completely identical to normal reproductive cells. The asexual reproductive cells of V. carteri cannot function directly as gametes but must be induced to cleave in the sexual developmental pathway to produce sexual spheroids with vegetative cells and sexual reproductive cells (Starr, 1970). A normal regA gonidium upon induction wlll cleave to form a sexual spheroid, but when the somatic cells regenerate in the presence of inducer they develop as eggs or sperm packets directly rather than cleaving in the sexual developmental pathways. In addition, a mutant of a regA strain has been isolated which does
234
DEVELOPMENTAL
BIOLOGY
not have gonidia but reproduces solely by somatic cell regeneration (Huskey, unpublished). Thus, at least one function necessary for normal reproductive cell formation which is missing in the gonidialess mutant is not required for somatic cell regeneration into reproductive cells. It has been suggested that the regA gene functions in early embryogenesis prior to the unequal cleavage since the sponteneous mutation rate to reg is similar to that of other mutations which must be expressed in the gonidium (kurn et al., 1978). Yet, the temperature shift experiments reported here show that the gene product acts in the somatic cells after cleavage stops. Taken together, these results suggest that either the primary gene transcript or the, presumably, protein product is present in the early embryo and is parcelled out to the somatic cells during cleavage. This hypothesis leads to possible explanations for the three phenotypic classes of regA mutants and the variable phenotype of the cold sensitive mutant. The class I mutants appear to be deficient in the reg gene product activity such that no cells contain the product or it does not act in any cell. The class II, polar mutants could have a problem related to the even distribution of the gene product such that large areas of the embryo are deficient while other areas may have an excess. The class III mutants could produce either a reduced amount of or a less efficient product, resulting in some cells establishing the differentiated state and others failing to do so. The variability of the cold-sensitive mutant could be explained using the assumptions that the gene product is parcelled out to the cells and that the lability of the gene product is reduced during cleavage compared to the time at which it acts. Under these assumptions, the earlier a shift is made to the restrictive temperature, the more likely is the inactivation of the gene product and the later shifts would have a lower level of gene product inactivation.
VOLUME
72, 1979
This would correlate with the more drastic phenotype of those spheroids which have a longer exposure to the restrictive temperature compared to the ones with a short exposure time. It has been suggested that cell size determines whether a cell will differentiate as a somatic cell or regenerate into a reproductive cell (Pall, 1975). This conclusion is based on the characteristics of mutants which have a premature cessation of division, resulting in large terminal cells and the apparent regeneration of these cells. The somatic cells of the polar and random regA mutants regenerate in one of two ways. Either the cells enlarge, take on the appearance of gonidia, and cleave or some of the cells along the line of demarcation between regenerating and differentiated cells begin to cleave without enlarging or changing appearance. Thus, a cell the size of a somatic cell can divide even though it does not enlarge above some minimum size. Also, the occurrence of intermediate-sized cells in the anterior region of the wild-type spheroid which do not develop into gonidia (Viamontes and Kirk, 1977) argues against cell size as the determining factor in the fate of cells. The technical assistance of P. 0. Cecil, S. Mumper, and G. Baran was greatly appreciated. This research was supported by NSF Grant BMS73-06742 to RJH; BEG was supported by NIH Training Grant 5TOl GM014.50. REFERENCES GRIFFIN, B. E., and HUSKEY, R. J. (1974). Genetic control of differentiation in Volvox. Genetics Ii’, ~27 (abstract). HUSKEY, R. J., GRIFFIN, B. E., CECIL, P. O., and CALLAHAN, A. M. (1979). A preliminary genetic investigation of Volvox carteri. Genetics 91, 229244. KOCHERT, G., and OLSON, L. W. (1970). Ultrastructure of Volvon carteri I. The asexual colony. Arch. Mikrobiol. 74, 19-30. KURN, N., COLB, M., and SHAPIRO, L. (1978). Spontaneous frequency of a developmental mutant in Volvox. Develop. Biol. 66, 266-269. MATAGNE, R. F. (1978). Fine structure of the arg-7 cistron in Chlamyomonas reinhardi. Mol. Gen. Genet. 160,95-99.
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PALL, M. L. (1975). Mutants of Volvox showing premature cessation of division: Evidence for a relationship between cell size and reproductive cell differentiation. In “Developmental Biology: Pattern Formation, Gene Regulation,” Vol. 2, ICN-UCLA Symposia on Molecular and Cellular Biology. W. A. Benjamin, New York. PICKETT-HEAPS, J. D. (1975). “Green Algae: Structure, Reproduction and Evolution in Selected Genera,” pp. 7-67. Sinauer Associates, Sunderland, Mass. SCOTT, W. A., MISHRA, N. C., and TATUM, E. L. (1973). Biochemical genetics of morphogenesis in Neurospora. Brookhaven Symp. Biol. 25,1-18. SESSOMS, A. H., and HUSKEY, R. J. (1973). Genetic control of development in Volvox: Isolation and
Somatic
Regenerator
characterization
Mutants
of morphogenetic
235 mutants. Proc.
Nat. Acad. Sci. USA 70,1335-1338. STARR, R. C. (1969). Structure, reproduction, and differentiation in Volvox carteri f nugariensis Iyengar, strains HK9 & 10. Arch. Protistenk. 111, 204-
222. STARR, R. C. (1970). Control of differentiation in Volvox. Develop. Biol. (Suppl.) 4, 59-100. STARR, R. C. (1975). Meiosis in Volvox carteri f. nagariensis. Arch. Protistenk. 117, 187-191.
SUZUKI, D. T. (1970). Temperature-sensitive mutations in Drosophila melanogaster. Science 170, 695-706. VIAMONTES, G. I., and KIRK, D. L. (1977). Cell Shape changes and the mechanism of inversion m Volvox. J. Cell Biol. 75, 719-730.
‘72, 226-235
BIOLOGY
(1979)
Genetic Control of Somatic Cell Differentiation Analysis
of Somatic
Regenerator
ROBERT J. HUSKEYANDBARBARA Department
of Biology,
Received
January
University
of Virginia,
25, 1979; accepted
in Volvox
Mutants E. GRIFFIN'
Charlottesville,
in revised
form
Virginia March
22901
30, 1979
The somatic regenerator (reg) mutants of Volvox carteri affect the ability of the normally terminally differentiated somatic cells to establish and/or maintain the differentiated state. Thirtynine reg mutants of four phenotypic classes have been mapped to two, unlinked genes, regA and regB. Mutants at the regA locus have one of three phenotypes: All somatic cells regenerate new spheroids, somatic cells in the spheroid posterior region regenerate while those in the anterior region differentiate as somatic cells, or regenerating and nonregenerating cells are randomly intermixed. The regB mutant has a random intermixture of regenerating and nonregenerating cells. Somatic cells regenerate new Volvon spheroids in two ways; the cells lose their characteristic shape, become immotile, enlarge and undergo cleavage similar to that of normal reproductive cells or undergo cell division without prior enlargement or loss of cell shape. Temperature shift experiments on a cold-sensitive reg mutant suggest that the gene product acts after the somatic cell initials are formed at the end of cleavage.
timing of the action of the gene product or products. We report here a genetic analysis of a number of somatic regenerator mutants along with evidence as to the time of action of the gene product.
INTRODUCTION
The somatic cells of the multicellular green alga, Voluox, have the characteristics of terminally differentiated cells. These cells have a characteristic shape, specialized organelles, and a characteristic pattern of proteins, and are not capable of cell division (Starr, 1969, 1970; D. Kirk and M. Kirk, personal communication). Mutants of VolVOX carteri have been reported in which the somatic cells are capable of reversing the differentiated state in that they lose the somatic cell characteristics, enlarge, and cleave to form new spheroids (Starr, 1970; Sessoms and Huskey, 1973; Griffin and Huskey, 1974; Huskey et al., 1979; Kurn et al., 1978). These somatic regenerator (reg) mutants appear to define a developmentally important gene or genes which regulate the establishment and/or maintenance of the differentiated state. Because of the significance of this developmental function, it is important to determine the number of genetic loci involved and the
MATERIALS
226 Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
METHODS
Culture conditions. The culture conditions, mutagenesis procedures, and crossing techniques are reported elsewhere (Huskey et al., 1979). All strains are derived from Volvox carteri f. nagariensis HKlO (female) and 69-lb (male), which were generously provided by R. C. Starr from the Algal Culture Collection at the University of Texas. Temperature shift experiments were performed with spot plate cultures using illuminated incubators (Precision Scientific) providing illumination from single, 15-W fluorescent strip lamps at a maximum of 1200 candles as measured by a Goosen Pan-Lux light meter; temperature was regulated at +l”C. Mutant isolation. The initial isolation of somatic regenerator (reg) mutants was by visual examination of a nitrosoguanidine-
I Deceased.
0012-1606/79/100226-10$02.00/O
AND
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mutagenized culture (Huskey et al., 1979). Spheroids with regenerating somatic cells are highly visible due to their dark green appearance which results from the enlarged cells on the surface of the spheroid. This technique has the disadvantage that recently released reg spheroids are difficult to distinguish from wild type so some mutants may be overlooked. Also, there is no enrichment or selection for the reg phenotype, which means that a great number of spheroids must be examined in order to find the mutants. A slight enrichment for reg and other developmental mutants can be effected by subjecting the mutagenized culture to phototactic selection prior to isolation of mutants. The reg spheroids are not capable of motion after the somatic cells begin regeneration so they do not respond to a phototactic stimulus. However, a number of morphogenetic mutants are also nonphototactic for a number of reasons; thus enrichment for reg mutants using photoactic selection is incomplete. It is possible to achieve a high selection efficiency for reg mutants using growth pattern on agar medium as the selective criterion. One plating method is to spread a mutagenized culture on the surface of agar medium. If the moisture content of the culture dish is high enough to avoid dehydration of the spheroids, clones of Volvox will arise on the agar. reg mutants give rise to dark green clones which can be seen on the uniform green background of the other clones. Another plating method is to spread the mutagenized culture on the surface of an agar plate using the soft-agar technique; reg clones are more compact than wild-type clones and can be removed from the plate for verification of phenotype (Kurn et al., 1978). Both of the plating techniques can be used in conjunction with a regimen to isolate temperature-conditional mutants by incubating the plates at high or low temperature. RESULTS
Phenotypes
of the reg mutants.
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young spheroids of all the reg mutants are nearly indistinguishable from wild type following inversion and release. However, as the reg spheroids begin to enlarge, some or all of the somatic cells begin to enlarge and lose their flagella, their eyespots usually disappear, and the production of extracellular matrix appears to slow. The number and location of the regenerating somatic cells constitute the basis for categorization of the reg mutants into four phenotypic classes. The class I mutants (33 isolates) show regeneration of all the somatic cells of the spheroids (Fig. 1). The regenerating cells enlarge and become vacuolated in a manner resembling developing gonidia. The regenerating cells rarely attain the size of a normal gonidium but they do go through a condensation of cytoplasmic material just prior to cleavage as do gonidia (Starr, 1969). The cleavage divisions of a regenerating cell are fewer in number than for a gonidium and the resulting spheroid has fewer reproductive and somatic cells than does a spheroid resulting from cleavage of a gonidium (Fig. 2). This class of mutant has been isolated by others, and two of these isolates are included in this study (Table 2). The class II reg mutants (3 isolates), which are similar to the one reported by Starr (1970)) have a polarization of regenerating cells such that the cells in the anterior portion and sometimes the extreme posterior region of the spheroid remain in the differentiated state while those cells in the posterior region undergo regeneration (Fig. 3). The somatic cells which do not regenerate occupy about one-quarter or less of the spheroid. Most of the regenerating cells of mutants in this class follow the pattern described for the class I mutants. However, some of the cells which occur at the junction of regenerating and nomegenerating cells undergo a different form of new spheroid formation. Instead of losing flagella and eyespots and enlarging to form a small, pseudogonidium, the cells begin to divide while retaining the characteristic
5
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FIG. 1. regA156 showing the class I phenotype. Some of the regenerating cells have cleaved and inverted. Daughter spheroids from the cleavage of gonidia can be seen in the interior of the parent spheroid. (The bars in Figs. 1, 3, and 5 represent LOO pm.1 FIG. 2. A spheroid resulting from cleavage and inversion of a regenerating somatic cell. Note the reduced number of gonidia (large cells in the interior) and somatic cells. (The bars in Figs. 2, 4, and 6 represent 10 pm.) FIG. 3. regA145 showing the class II or polar phenotype. The clustered, enlarged cells on the surface of the spheroid are beginning regeneration. The gonidia can be seen as large, darker cells in the interior of the spheroid. FIG. 4. Surface view of a regA of class II phenotype. Nonregenerating somatic cells (SC) are present. Some cells have enlarged and undergone cleavage (cl) to form small embryos (indicated by arrow) or inverted to form small spheroids complete with gonidial initials (to right of indicated embryo). Some cells regenerate by direct division (d) to form a spheroid containing only somatic cells (see Fig. 6). Daughter spheroids resulting from normal gonidial development can be seen underneath the surface cell layer. FIG. 5. regA141 showing the class III or random phenotype with an intermixture of somatic cells and enlarged, regenerating cells. FIG. 6. A spheroid resulting from division of a regenerating somatic cell without prior enlargement (compare to d of Fig. 4). Gonidia are not present. 228
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shape of somatic cells (Fig. 4). Each daughter cell has two flagella, but only one of the cells of the growing individual contains an eyespot. When division ceases, the resultant spheroid contains 20-50 cells, but few if any obvious reporductive cells are present in the interior of the spheroid (Fig. 6). These apparently sterile spheroids, which resemble the palmella stage of Chlamydomonas or the adult forms of other colonial members of the Volvo&es (PickettHeaps, 1975), usually develop reproductive cells resembling gonidia or regenerating somatic cells. Mutants of the class III type (2 isolates) have a near-equal number of regenerating and nonregenerating cells, with a random, intermixed distribution of the two cell types (Fig. 5). The cells regenerate in the patterns of the class II type. The class IV reg mutant resembles the class III mutants with an intermixture of regenerating and nonregenerating cells, but regeneration occurs much earlier in the life cycle and the nonregenerating cells appear not to fully differentiate. For example, the spheroids are motile for a very short time and very little spheroid expansion is evident, suggesting that there is little production of extracellular matrix. The class IV mutant is very sensitive to agitation and breaks up into single cells in an aerated culture. Genetic analysis of reg mutants. The only diploid phase of the Volvox life cycle is the zygote which germinates by meiosis to yield one haploid zoospore (Starr, 1975). Therefore, strains carrying single site mutations should show a 1:l segregation of progeny when crossed to wild type; crosses of allelic mutants will give all mutant progeny unless intragenic recombination occurs; crosses of unlinked mutants of similar phenotype should result in a 3: 1 ratio of mutant to wild-type progeny; and crosses of mutants in linked genes will result in a mutant: wild-type ratio between the two extremes (Huskey et al., 1979). All of the reg mutants behave as single
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Mutants
gene mutants in crosses with wild-type, showing a 1:l segregation of reg to wild type (Table 1). A similar finding was reported by Starr (1970) with a reg mutant which is included in this study, regA154. These results mean that none of the reg mutants reported here are due to multiple TABLE ONE-FACTOR reg parent
regAlO1 regA102 regA 103 regA104 regA 105 regAlO8 regAlO7 regA 108 regA 109 regAll1 regAll2 regAll3 regAl14 regA115 regAl16 regAl17 regAll8 regAll9 regA123 regA125 regA126 regA128 regA135 regA137 regA138 regAl41 regA142 regA145 regA147 regA149 regA150 regA 153 regA154 regA155 regA156 regA157 regA159 regAl60 regB144
Number
1 CROSSES” ofprogeny
p (>)
reg
Wild
Total
73 64 64 54 179 63 62 47 64 58 96 46 55 79 60 57 92 65 84 85 148 138 52 74 45 40 58 54 284 133 67 101 84 122 67 87 15 52 328
57 59 54 54 184 57 61 52 71 49 106 50 45 68 56 54 96 58 77 90 151 109 51 62 44 48 59 53 277 154 50 95 80 99 51 65 18 57 294
130 123 118 108 363 120 123 99 135 107 202 96 109 147 116 111 188 123 161 175 299 247 103 136 89 88 117 107 561 287 117 196 164 221 118 152 33 109 622
0.10 0.60 0.30 0.90 0.70 0.50 0.90 0.60 0.50 0.30 0.40 0.60 0.30 0.30 0.70 0.70 0.70 0.50 0.50 0.70 0.80 0.05 0.90 0.30 0.90 0.30 0.90 0.90 0.90 0.20 0.10 0.60 0.70 0.10 0.10 0.05 0.60 0.60 0.10
n In each cross the reg mutant was the female strain and was crossed to a wild-type male strain. The deviation of the progeny classes from the expected 1:l ratio was compared to the chi-squared distribution.
230
DEVELOPMENTAL
mutations genes. Pairwise performed which had type male the reg by
in unlinked
or loosely
BIOLOGY
linked
crosses of the reg mutants were using six separate reg mutants been recovered in the mating(mtm) strain (Table 2). In 57 of reg crosses, the progeny were all TABLE TWO-FACTOR
mtf
Cross
VOLUME
mutant with the exception of one wild-type recombinant. These results suggest that this set of mutants is allelic at the same genetic locus, which has been designated regA. The regA group includes all the mutants of the class I, II, and III phenotypes (Table 2). 2
CROSSES OF
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reg MUTANTS”
Cross
2
Cross
3
Parent
mtm
mtm
Progeny
Parent
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reg
+
mtm
Progeny
Parent
Progeny
Parent
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reg
+
reg
+
regAlO1 regAll2 134 0 regA102 regAll2 0 regAlO5” 286 32 0 regAl41 b 46 1 regA 103 regAll2 407 0 regAlO4 regAlO1 83 0 regAll2 755 0 regAl41’ 85 0 regAlO5” 0 regAlO1 37 regAll1 23 0 regAl41 b 35 0 regAlO6 regAll2 403 0 regA107 regAlO1 0 regAl41 b 48 44 0 regAlO8 regAlO1 34 0 regAll2 97 0 regAlO9 regAlO1 7 0 regAll2 586 0 regAl41’ 24 0 regAll1 regAlO1 56 0 regAlO5” 9 0 regAl12 regAl42” 80 0 regAl41 b 69 0 regAll3 0 regAl41 b regAlI 345 18 0 regAll4 regAll2 0 173 regAll5 regAlO1 239 0 regAll2 242 0 regAl41’ 153 0 regAll6 0 regAl41’ regAll2 533 6 0 regAl17 regAlO1 153 0 regAll2 191 0 regAll8 regAll2 73 0 regAll9 regAlO1 80 0 regAll2 123 0 regAl23’ 0 regAll2 88 regA125 regAll2 322 0 0 regA126 regAl45” 138 regA128 regAll2 539 0 regAl41 b 31 0 regA135 regAll2 213 0 regAl41 b 205 0 regA137 regAll2 4 0 regAl41 b 75 0 regA138 regAll2 81 0 regA142” regAl41 b 148 0 regA147 regAll2 320 0 regA149 regAlO1 83 0 regA150 regAl45” 71 0 regAl41 b 0 regA153 67 regA154d regAl41’ 273 0 regAl55’ regA 149 422 0 regA156, regAll2 185 0 regA157 regAl41’ 0 154 regA159 regAll2 0 85 regAlGO regA145 171 0 regB144’ regAll2 61 24 o All regA mutants are of the class I phenotype, with the following exceptions: “regAlO5, regA123, and regA145 are mutants of class II phenotype; hregA141 and regA142 are mutants of class III phenotype; ‘regBl44 is the only class IV mutant; ‘isolated as a spontaneous mutant by R. C. Starr; ‘isolated induced mutant by D. L. Kirk; ‘isolated as a spontaneous mutant by P. O’Farrell.
as a nitrosoguanidine-
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type. When the mutant is grown at the permissive temperature (37°C) and shifted to the restrictive temperature (24”C), the resulting spheroids will be mutant in phenotype if the shift occurs before the time period in which the gene product acts (the sensitive period) or they will be wild-type in phenotype if the shift occurs after the sensitive period (Suzuki, 1970). Alternatively, when the mutant is grown at the restrictive temperature and shifted to the permissive temperature, the resulting spheroids will have a wild-type phenotype if the shift is made before the sensitive period or a mutant phenotype if the shift is made after the sensitive period. The temperature shift experiments on regAlSO were complicated by two factors. First, the incubation conditions affect the generation time of wild-type as well as reg mutants. For regA150, the generation time is 48 hr when grown at 37°C or 196 hr when grown at 22°C. This fourfold elongation of the life cycle is nearly uniform for all parts of the cycle and may result from a combination of lowered temperature and slightly lowered light intensity due to differences in the illuminated incubators used. For this reason, the data presented in Fig. 7 are
The reg mutant of class IV phenotype gave a 3:l segregation of reg to wild-type when crossed with one of the regA mutants, which means that this mutant defines a second, unlinked locus, regB. Previously reported data indicate that the regA locus is not linked to the mating type locus (mt) or any other of the existing mutants of Volvox carteri (Huskey et al., 1979). Based on these results, the regA locus has been assigned to linkage group II of Voluox. The regB mutant is also unlinked to mt and segregates independently with markers from linkage groups II, III, V, VII, and VIII (data not presented). Temperature shift experiments. One of the reg mutants, regAlSO, is a cold-sensitive mutant which has the mutant phenotype when grown at 24°C but has a wildtype phenotype when grown at 37°C. This mutant strain was used in temperature shift experiments in an attempt to determine the time of action of the gene product. The basic experiment is to grow the mutant synchronously at one temperature and shift to the other temperature condition at various times after the initiation of gonidial cleavage. The resultant spheroids are scored for mutant versus wild-type phenoTIMING
OF THE
LIFE CYCLE
(hrs 1
6
I2
I6
24
30
24
46
72
96
120
OF THE LIFE
CYCLE
100
shift down (33OC) 42 46 rhlft “P (24-Z) 144 I66 I96 4 (shift up. Eg:(I)
36
(shift ,750 FRACTION
231
down,w_t) ,675
FIG. 7. Temperature shift experiments on regAl5f.T”. Gonidia from regAl5F were isolated and cultured in illuminated incubators at either 24°C (A) or 33°C (0). At the times indicated, samples were shifted to the other incubation temperature and the resulting spheroids were scored for somatic cell regeneration. A spheroid was scored as a regenerator if a sector of regenerating cells was present. The generation times for Volvox grown at the two temperatures are given at the top of the figure. The data are plotted as the fraction of the life cycle for the temperature of the original culture.
232
DEVELOPMENTAL BIOLOGY
plotted as the fraction of the life cycle rather than in hours and are based on the initial temperature of incubation. Furthermore, the beginning of the experiment is based on the initiation of cleavage of gonidia since this is a portion of the life cycle which is reproducibly identifiable in both reg and wild-type spheroids. The second problem in analyzing the temperature shift experiments is that the number of regenerating somatic cells in any one spheroid varied as a function of the length of time at the restrictive temperature. Thus, spheroids shifted to the restrictive temperature at early times prior to the sensitive period had more regenerating cells than those shifted just before the sensitive period. The reciprocal result occurred with the shifts to the permissive temperature. Because it was difficult to quantify the amount of regeneration in any one spheroid, the spheroids were scored as reg in phenotype if they had a sector of regenerating somatic cells. It should be noted here that the regAlSO spheroids show three classesof regenerator phenotype depending on the time of the shift to the restrictive temperature. Spheroids which result from early shifts to the restrictive temperature have the class II or polarized phenotype, while spheroids which result from late shifts prior to the sensitive period have the class III or random phenotype. Spheroids which result from continued growth at the restrictive temperature have the class I or complete phenotype. Thus, the one mutant strain, regAlSO, mimics all three classesof regenerator mutants mapping to the regA locus depending on the length of time at the restrictive temperature. This supports the results of the genetic analysis that the regA locus is a single gene. The results of the temperature shift experiments (Fig. 7) indicate that the sensitive period for the action of the reg gene product begins following the end of cleavage and inversion (one-fourth of the life cycle) and ends following release of the young spheroids (one-half of the life cycle).
VOLUME 72, 1979
It is during this time period that the cells develop eyespots, the characteristic cell shape becomes apparent, the cells begin synthesis of the extracellular matrix, and the chloroplast becomes morphologically distinct (Kochert and Olson, 1970). Thus, the regA gene product appears to function at the time when somatic cells begin to display a number of the characteristics of the differentiated state. An important result of the temperature shift experiments is that a regA150 embryo or spheroid which is shifted after the sensitive period does not show regeneration of the somatic cells; that is, there is a definite sensitive period. Only the somatic cells of the spheroids of the next generation show regeneration and only if maintained at the restrictive temperature. This result suggests that the reg gene product is required for the initial establishment of a stably differentiated state but is not required continuously for the maintenance of that state. When the temperature shift experiments with regA150 were performed elsewhere under conditions resembling those reported here, similar results were obtained. However, it was also observed that if incubation at both permissive and restrictive temperatures was carried out at the same high (about 1850-ft-candle) level of illumination, the frequency of expression of the regenerator phenotype was reduced (D. L. Kirk, personal communication). The significance of this result is being explored further, but it appears to indicate that the light level in some way modulates the expression of the temperature-conditional phenotype in the regAlSO mutant. DISCUSSION
The 39 independently isolated reg mutants reported here define only two, unlinked loci of the Volvox genome-regA and regB. This fact reinforces the original suggestion that the reg mutants identified a genetic function which is critical for differentiation (Starr, 1970). This is in contrast to findings in some organisms such as Neu-
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rospora where mutants of similar morphological phenotype have been found to affect the enzymes of basic biochemical pathways and occur in a number of different structural genes rather than in a few developmentally important regulatory genes (Scott et al., 1973). The limited number of reg loci also lends more credence to the basis for the spontaneous mutation rate method used by Kurn et al. (1978) to suggest that the reg gene functions in the young embryo rather than in the somatic cell. The occurrence of three phenotypic variants among mutants mapping at the regA locus is suggestive of a set of closely linked genes rather than a single gene. However, two results offer evidence that the regA locus is a single gene. First, in 57 pairwise crosses of regA mutants with each other, only one wild-type recombinant was recovered in over 9000 total progeny. In the closely related organism, Chlamydomonas, intragenic recombination occurs at a level of 1% or less (Matagne, 1978). Since recombination at the regA locus is less than l%, it can be tentatively identified as a single gene using a recombinational definition. Second, the temperature-conditional mutant, which is probably due to a missense mutation, mimics the three phenotypic classes of regA mutants depending on the length of developmental time at the restrictive temperature. It is easiest to explain this behavior if only one gene is involved. Furthermore, the fact that a temperature-conditional mutant was isolated means that a gene product is made and it is likely that the ultimate gene product is a protein. The temperature shift experiments on the cold-sensitive mutant establish the time period in which the gene product acts. This period begins after cessation of cleavage, meaning that each somatic cell is probably acting independently in establishing the stably differentiated state. This also means that the unequal division which normally sets somatic and gonidial initials apart during cleavage (Starr, 1969) is not the only point at which control is exerted over the
233
reproductive potential of somatic cells. In addition, the reg gene product is not required continuously to maintain the differentiated state since the shift experiments show an end point to the sensitive period. Thus, the regA gene product acts in the somatic cell initials to establish the stable, differentiated state and, apparently, is not required for maintenance of differentiation. The suggestion that the somatic cells of reg mutants undergo dedifferentiation rather than failing to establish the differentiated state is based on the observation that somatic cells of reg mutants have the general morphology of fully differentiated somatic cells and are therefore fully differentiated. In fact, preliminary biochemical studies favor this notion of full differentiation in regA somatic cells in that these cells produce the same malate dehydrogenase isozyme seen only in wild-type somatic vegetative cells and distinct from the isozyme of gonidia (K. V. Chace, personal communication). However, the finding that some regenerating cells divide without prior enlargement and loss of characteristic shape means that some caution must be used in defining the differentiated state of Volvox vegetative cells. In addition, this finding is very significant in opening up the study of the evolution of the Volvocales. This latter point will be explored in more detail in a subsequent publication. The regenerating somatic cells of regA mutants are not completely identical to normal reproductive cells. The asexual reproductive cells of V. carteri cannot function directly as gametes but must be induced to cleave in the sexual developmental pathway to produce sexual spheroids with vegetative cells and sexual reproductive cells (Starr, 1970). A normal regA gonidium upon induction wlll cleave to form a sexual spheroid, but when the somatic cells regenerate in the presence of inducer they develop as eggs or sperm packets directly rather than cleaving in the sexual developmental pathways. In addition, a mutant of a regA strain has been isolated which does
234
DEVELOPMENTAL
BIOLOGY
not have gonidia but reproduces solely by somatic cell regeneration (Huskey, unpublished). Thus, at least one function necessary for normal reproductive cell formation which is missing in the gonidialess mutant is not required for somatic cell regeneration into reproductive cells. It has been suggested that the regA gene functions in early embryogenesis prior to the unequal cleavage since the sponteneous mutation rate to reg is similar to that of other mutations which must be expressed in the gonidium (kurn et al., 1978). Yet, the temperature shift experiments reported here show that the gene product acts in the somatic cells after cleavage stops. Taken together, these results suggest that either the primary gene transcript or the, presumably, protein product is present in the early embryo and is parcelled out to the somatic cells during cleavage. This hypothesis leads to possible explanations for the three phenotypic classes of regA mutants and the variable phenotype of the cold sensitive mutant. The class I mutants appear to be deficient in the reg gene product activity such that no cells contain the product or it does not act in any cell. The class II, polar mutants could have a problem related to the even distribution of the gene product such that large areas of the embryo are deficient while other areas may have an excess. The class III mutants could produce either a reduced amount of or a less efficient product, resulting in some cells establishing the differentiated state and others failing to do so. The variability of the cold-sensitive mutant could be explained using the assumptions that the gene product is parcelled out to the cells and that the lability of the gene product is reduced during cleavage compared to the time at which it acts. Under these assumptions, the earlier a shift is made to the restrictive temperature, the more likely is the inactivation of the gene product and the later shifts would have a lower level of gene product inactivation.
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This would correlate with the more drastic phenotype of those spheroids which have a longer exposure to the restrictive temperature compared to the ones with a short exposure time. It has been suggested that cell size determines whether a cell will differentiate as a somatic cell or regenerate into a reproductive cell (Pall, 1975). This conclusion is based on the characteristics of mutants which have a premature cessation of division, resulting in large terminal cells and the apparent regeneration of these cells. The somatic cells of the polar and random regA mutants regenerate in one of two ways. Either the cells enlarge, take on the appearance of gonidia, and cleave or some of the cells along the line of demarcation between regenerating and differentiated cells begin to cleave without enlarging or changing appearance. Thus, a cell the size of a somatic cell can divide even though it does not enlarge above some minimum size. Also, the occurrence of intermediate-sized cells in the anterior region of the wild-type spheroid which do not develop into gonidia (Viamontes and Kirk, 1977) argues against cell size as the determining factor in the fate of cells. The technical assistance of P. 0. Cecil, S. Mumper, and G. Baran was greatly appreciated. This research was supported by NSF Grant BMS73-06742 to RJH; BEG was supported by NIH Training Grant 5TOl GM014.50. REFERENCES GRIFFIN, B. E., and HUSKEY, R. J. (1974). Genetic control of differentiation in Volvox. Genetics Ii’, ~27 (abstract). HUSKEY, R. J., GRIFFIN, B. E., CECIL, P. O., and CALLAHAN, A. M. (1979). A preliminary genetic investigation of Volvox carteri. Genetics 91, 229244. KOCHERT, G., and OLSON, L. W. (1970). Ultrastructure of Volvon carteri I. The asexual colony. Arch. Mikrobiol. 74, 19-30. KURN, N., COLB, M., and SHAPIRO, L. (1978). Spontaneous frequency of a developmental mutant in Volvox. Develop. Biol. 66, 266-269. MATAGNE, R. F. (1978). Fine structure of the arg-7 cistron in Chlamyomonas reinhardi. Mol. Gen. Genet. 160,95-99.
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PALL, M. L. (1975). Mutants of Volvox showing premature cessation of division: Evidence for a relationship between cell size and reproductive cell differentiation. In “Developmental Biology: Pattern Formation, Gene Regulation,” Vol. 2, ICN-UCLA Symposia on Molecular and Cellular Biology. W. A. Benjamin, New York. PICKETT-HEAPS, J. D. (1975). “Green Algae: Structure, Reproduction and Evolution in Selected Genera,” pp. 7-67. Sinauer Associates, Sunderland, Mass. SCOTT, W. A., MISHRA, N. C., and TATUM, E. L. (1973). Biochemical genetics of morphogenesis in Neurospora. Brookhaven Symp. Biol. 25,1-18. SESSOMS, A. H., and HUSKEY, R. J. (1973). Genetic control of development in Volvox: Isolation and
Somatic
Regenerator
characterization
Mutants
of morphogenetic
235 mutants. Proc.
Nat. Acad. Sci. USA 70,1335-1338. STARR, R. C. (1969). Structure, reproduction, and differentiation in Volvox carteri f nugariensis Iyengar, strains HK9 & 10. Arch. Protistenk. 111, 204-
222. STARR, R. C. (1970). Control of differentiation in Volvox. Develop. Biol. (Suppl.) 4, 59-100. STARR, R. C. (1975). Meiosis in Volvox carteri f. nagariensis. Arch. Protistenk. 117, 187-191.
SUZUKI, D. T. (1970). Temperature-sensitive mutations in Drosophila melanogaster. Science 170, 695-706. VIAMONTES, G. I., and KIRK, D. L. (1977). Cell Shape changes and the mechanism of inversion m Volvox. J. Cell Biol. 75, 719-730.