Download genetic control of pigment differentiation in somatic cells

AM. ZOOLOCIST, 3:57-69 (1963).
GENETIC CONTROL OF PIGMENT DIFFERENTIATION
IN SOMATIC CELLS
WILLIAM K. BAKER
Department of Zoology, University of Chicago
The process of cellular differentiation
may be studied by using the techniques
of a variety of biological disciplines. Embryologists may approach problems of induction, cell recognition and contact,
induced enzyme synthesis, etc. through use
of biochemical or biophysical techniques,
but often the powerful tool of genetic
analysis is left unused. One facet of this
problem which might be particularly susceptible to analysis by use of genetic techniques is implicit in the title of this paper:
the genetic control of pigment differentiation. By the term "genetic control" it is
meant to imply that not only is the process
of cellular differentiation a species specific
one and determined by the individual's
own genetic constitution, but also in most
cases the cells of a tissue which have differentiated in a similar fashion are related
by descent and thus the process is one of
somatic cell genetics—using the term "genetics" is its broad sense.
Let us focus our attention on the latter
process and ask the following question:
what would be an ideal system for studying the genetic control of somatic cell differentiation? As an elementary system for
such studies, one might pick a tissue in
which all of its cells were of the same
cellular type and related by descent; and
in addition, one would want a tissue in
which certain of the cells performed one
function, whereas the remaining cells
either did not perform this function or
performed another. If this differentiation
of function was under genetic control, and
if the investigator had some way of altering this control, then the requirements of
this ideal system might be met. I should
hasten to add that in such a system one is
not studying the somatic differentiation of
different cellular types, but a much simMuch oC the research reported herein has been
supported by Grant Xo. RG-7428 from the U. S.
Public Health Service.
pier process—the differentiation of somatic
cell function. It seems reasonable to conclude that any information gathered on
functional differentiation might be of
prime importance in unravelling the complex series of events leading to cellular
differentiation.
There is a long-known genetic phenomenon which fulfills many of the prerequisites of this ideal test system; it is called
position-effect variegation. Schultz (1941)
was among the first to recognize the potentialities of variegation in Drosophila as
a system for studying development. More
recently Becker (1960) and Baker (1959,
I960) have emphasized this view. Although
a lengthy discussion of the genetic factors
causing this variegation is not essential to
the subject under discussion, let us recall
certain of the characteristics of this phenomenon. The somatic variegation caused
by position effect is almost invariably associated with a chromosomal aberration,
one of whose break points is close to the
gene whose expression is affected. Another
necessary requirement is that one of the
breaks of the aberration must be in a
heterochromatic region of a chromosome.
It appears that there are three sufficient
conditions which will invoke the variegation: (1) If a gene is normally located in
a euchromatic region, it may show variegated expression when placed, by the
rearrangement, near a heterochromatic region. (2) If the locus is normally in a
heterochromatic region, the gene expression may be affected by relocation to a
euchromatic one, or (3) by placing it near
a foreign heterochromatic region. The
cases of position-effect variegation that
will be discussed in this paper are all
recessive; i.e., a rearrangement, R, fulfilling the above conditions and containing
the dominant wild-type allele of a gene,
say R(w+), will only produce variegated
(57)
58
WILLIAM K. BAKER
flies when it is heterozygous with a recessive mutant allele of the gene or when the
flies are homozygous for the rearrangement. Thus variegation is displayed in
two genotypes: R(w + )/?n and R (w+)/
R(zu+), but not in R(w+)/iv+ or R (w)/
w+.
It appears that this type of somatic variegation may be observed in the expression of any gene whose mutant effect
covers sufficient somatic tissue to allow
detection by the observer. One of the
favorite objects for study has been the
pigmentation in the compound eye. The
compound eye of D. melanogaster is composed of about 800 individual ommatidia
or facets and each facet is composed of
about 30 cells. Over 20 cells within or
associated with an ommatidium may form
pigment, and these cells are distributed
over four different regions. At first glance
it might seem that such a complicated
organ would fall so far short of our idealized system for studying cellular differentiation of function that it would be useless.
However, as we shall see later, each facet
may be treated as a unit and the question
asked, does it or does it not produce any
pigment? It is also possible to get an answer to the question, does one group of
facets contain pigment visibly different
from another group?
A meaningful discussion of the mechanisms for the genetic control of this
variegation must be placed within the
framework of Drosophila development. It
is important to remember that every stage
of the life cycle of an organism is under
some genetic control. Whether the activities of a group of cells are under the
guidance of their own nuclear genes or
whether they are under the direction of
genes of the previous generation depends
on the particular stage of the life cycle
being examined.
Figure 1 represents a diagrammatic view
of the general ontogeny of D. melanogaster. It is well to recall the sets of different
genetic instructions that are present in the
nuclear material of unfertilized and of
fertilized eggs. The sets of genetic infor-
mation in the female pronucleus, e<., and
in each of the three polar body nuclei, ei,
en, and es, may be of two types for any one
gene. At the third cleavage division the
two posteriorly located polar body nuclei
fuse together and at the sixth division,
these two fuse with the anteriorly located
polar body nucleus. Subsequently at the
ninth division the chromosomes in this
triploid nucleus begin to fragment and
disappear (Rabinowitz, 1941). Because of
this chromosomal disintegration during
formation of the blastema, polar body
nuclei are not thought to take any part in
normal development (Rabinowitz, loc. cit.;
Sonnenblick, 1950); however, note that no
experimental evidence bearing on the correctness of this thought is at hand. Aside
from genetic information in nuclear material within the unfertilized egg, the cytoplasm of this cell is, of course, genetically
determined by the genotype of the mother,
m. Material synthesized in the nurse cells
(and follicle cells as well) is "poured"
(King, 1956) into the oocyte at the border
between it and the nurse cells. Since the
unfertilized egg contains a wide variety of
polynucleotide fragments of RNA (Levenbok, Travaglini, and Schultz, 1953), there
is the possibility that the ooplasm contains
not only products synthesized according to
genetic instructions of maternal origin, but
also contains the genetic information to
direct further syntheses specified by these
instructions.
Fertilization of the egg brings into this
cell other sets of divergent genetic information. The sperm pronucleus carries its
set of instructions, s», and one must
consider the possibility that cytoplasmic
material synthesized by the father or cytoplasmic material capable of carrying genetic information may enter the egg
through the sperm. This information, p,
would be specified by the paternal genotype.1 Thus we see that there would be a
1
The possibility of divergent information
through pohsperm) need not be considered in
\ieu of the e\idence (unpublished) recently obtained b\ Dr. P. E. Hildreth and substantiated by
Dr. Sheila J. Counce which indicates that polys])crm\ K a rare event in D. mrianogastrr.
GENETIC CONTROL OF DIFFERENTIATION
. ^
UNFERTILIZ
FERTILIZED
59
EGG
polar body*.--1
nuclei
pronucleus
dpronucleus
cytopiqsmic
stratification
produced by
maternal
genotype
V
possible paternal
cytoplasm entering with sperm
I
22-24 hours
3 r d INSTAR
2nd
I st
INSTAR
23 hours
,25 hours
eye pigment formation
49
hours
INSTAR
IMAGO
47 hours
FIG. 1. Information content of eggs and general development of Drosofrhila.
maximum of five possibly divergent sets
of instructions which might be available
for the developing embryo to "read." Any
of these sets of instructions might be capable of influencing processes at the very
earliest stages of ontogeny, but the sets
which could be of importance to later
development would be those either present
in large amounts at fertilization or those
instructions capable of replication.
Since it is the pigmentation of the compound eye with which we shall be primarily concerned, a few remarks about its
development are pertinent. The paired
imaginal discs which will give rise to the
eye, the antenna, and part of the head
hypodermis are already present when the
larva hatches from the egg. They have
arisen as outpocketings of ectoderm from
the dorsal wall of the pharyngeal cavity
(see review by Bodenstein, 1950). As de-
velopment proceeds, the eye and antenna
portions of the disc become segregated. In
the mature third instar larva, the eye disc
consists of clusters of four cells. These
clusters are arranged in regular rows and
their number corresponds approximately
to the final number of ommatidia. The
eye discs evaginate about 12 hours after
pupation, and during the pupal period the
final differentiation and specialization of
the eye takes place. It is important to note
that the first signs of pigment in the eye
appear about two days after pupation, approximately midway in the pupal period.
Let us return from this brief sketch of
Drosophila development to a reconsideration of the experimental approach for
studying differential cell function. Much
of the work on position-effect variegation
in our laboratory has used a rearrangement
involving the white region located on the
60
WILLIAM K. BAKER
VARIEGATED
yw
MALES
VARIEGATED
FEMALES
f
•H-
yt
ywf YL- Y^scP-Y; D p A
y w / s c 8 • Y ; Dp/+
F[G. 2. Cytological and genetic constitution of
flies showing white variegation caused by position
effect of Dp (rum)264-58a.
X chromosome of D. melanogaster. This
rearrangement, Dp (w"*)264-58a, is an insertion of about 20 salivary chromosome
bands into the proximal heterochromatic
region of the left arm of the third chromosome. It is pictured in Fig. 2. Along with
the white (w) locus are included the genes
roughest (rst), facet (fa), facet-notch (fa"),
facel-notchoid (fan°), split (spl), notchoid
(nd), and diminutive (dm). Most of the
flies whose somatic variegation we shall
discuss carry the duplication heterozygously on the third chromosome and are either
homozygous or hemizygous for the mutant
white on the X. In addition the males
carry an X chromosome to which the fertility factors of the Y chromosome have
been attached and the females carry attached-X chromosomes. The genotypes are
also shown in Fig. 2.
Figure 3b-g shows typical examples of
white variegation caused by position effect
in Drosophila. These examples of the pigmentation patterns produced in the ventral two-thirds of the eye were selected
from a sample of several hundred eyes
drawn. The patterns illustrated in this
figure were picked to show some of the
common types observed and to indicate
that positive and negative images of the
same pattern were detected. It is the information derived from these patterns
which establishes the relevance of this va-
riegation to problems of differential cell
function. Upon examination of variegated
patterns, one is impressed immediately
with three facts, (i) The pigmented or
nonpigmented areas are, in general, continuous in nature; i.e., there is not a
random salt-and-pepper distribution of
pigment within the eye. (ii) One can find
several sorts of patterns that are often repeated among the variegated eyes. (Hi)
Both positive and negative images of the
same pattern are observed. Taken together,
these three factors suggest that the pigmentation pattern might be clue to events
that took place during ontogeny of the
compound eye, the results of these events
being replicated in the cells which gave
rise to a given group of ommatidia.
The elegant experiments of Becker
(1957), in which he traced the ommatidial
lineage, convert this suggestion into a fact.
Becker studied the twin spots (w/w and
wco/wc° tissue) produced by somatic crossing over in w/w" females. Somatic crossing over was induced by X-irradiating
these females when they were first instar
larvae. Figure 4 illustrates how this genetic
technique can be utilized to determine
cell lineage. Analysis of the patterns pro-
d.
FIG. 3 a.—Areas of common cell lineage (I-VIII)
in \entral half of compound e\e, after Becker
(1957). b.-g.—Patterns of variegation caused byposition effect. See text.
GENETIC CONTROL OF DIFFERENTIATION
w
w co
wco
—)
0
TWIN
SPOT
FIG. 4. Becker's scheme (1957) for determining
cell lineage in the compound eye through induced
somatic exchange. The shaded circles indicate cells
that will produce the intermediate phenotype of
w/zv"0, open circles the to phenotype, and closed
circles the wco phenotype. The induced crossover
is shown to occur at the second round of cell division within this clone.
duced by twin spots indicated that at the
end of the first larval instar there are
present in each eye disc about eight cells
whose descendants will form the ommatidia and part of the hypodermis of the
ventral one-half of the head. The cell lineage is relatively regular in the ventral half
making it possible to delineate the size
and location of the eight sectors of ommatidia arising from these eight cells. In Fig.
3a these sectors are pictured diagrammatically.
This beautiful analysis by Becker can
thus provide a basis for analysis of the
question as to whether or not the patterns
of pigmentation found in position-effect
variegation have a cell lineage basis. It is
certain that many, if not most, of the patterns have such a basis, as can be readily
seen by comparing the patterns pictured in
Fig. 3b-g with the diagram outlining the
sectors of common cell lineage Fig. 3a.
61
For example, in pair b and c the entire
sector I is affected; in pair d and e sectors
IV, V, VI and VII are alike; and in pair
f and g sectors I, VI, VII, and VIII are
affected. Thus, in many cases, these patterns cover entire sectors or even more
than one sector. This result means that in
many cases it is during the first larval instar that a decision is made whether one
of the eight cells and. its descendants will
or will not produce pigment. You will
recall that it is much later—seven days
after this time—that the first evidence of
pigment appears in the eye.
Since one is able to find among the
variegated eyes positive and negative images of the same pattern, it is apparent
that cells which will form a given sector
are not rigidly predisposed either to produce or not to produce pigment. It is
possible, although a quantitative study has
not been made, that this is a stochastic
process superimposed on a gradient across
the eye.
The cell-lineage nature of the variegation and its early and rather rigid determination means that position-effect
variegation can be viewed as a problem of
somatic cell genetics. The clonal nature
of the patterns, at least at first sight, seems
to add weight to the hypothesis that variegation is the result of somatic mutation.
Let us look at the evidence bearing on
whether variegation is caused by a change
at the gene level of organization. Five
lines of evidence against this assumption
will be marshalled from data on white
variegation and light variegation, (//, =
light, a gene located in the proximal heterochromatic region of the left arm of
chromosome 2).
1) If position-effect variegation were the
result of somatic mutation; i.e., R(ii>+)
—»R (w) in a white variegated eye or
R (//+)-» R (It) in a light-variegated eye
then the areas of wild-type pigmentation
in the eye would be R(iu+)/iu or R (//+)/
// and the mutant regions R (w)/w or
R (lt)/ll. Now, it has been known for some
time (Gowen and Gay, 1933, 1934;
Schultz, 1936) that extra Y chromosomes
62
WILLIAM K. BAKER
in the genome produce more pigmentation
in white-variegated flies, whereas the removal of Y chromosomes causes more of
the eye to lack pigment. Interestingly,
Schultz (1936) first showed that the effect
of Y chromosomes is exactly the reverse
with position-effect variegation involving
the light locus. We (Baker and Rein,
1962) have recently obtained more critical
evidence on this pojnt by studying quantitatively the effect of six different Y
chromosomes, or fragments thereof, on
variegation of light and on two different
states of white variegation. The experiments were designed such that—by use of
successive matings of an individual maleone and the same Y fragment was introduced into each of the three systems and
a comparison made inter se. The results
are quite clear. If one arranges the Y
fragments in order of their ability to enhance pigmentation in the light-variegated
system, the order is exactly reversed in the
two white systems. Now, if Y chromosomes were changing the frequency of
somatic mutation, this would mean that
one and the same Y fragment was increasing mutation frequency in the white system and decreasing it in light-variegated
eyes. There is no precedent for such a
clichotomous mutagenic action of the same
agent. An alternative interpretation, based
on timing of the postulated mutational
event, remains. One might imagine that a
Y chromosome fragment in the whitevariegated system allowed the postulated
mutations to occur at a later stage of development of the eye than in the light
system, thus producing smaller areas of
mutant tissue in the former system than in
the latter (see below, however).
2) It has been shown unequivocally
(e.g., Gowen and Gay, 1933b, 1934) that
when flies showing position-effect variegation were raised at a low temperature, the
amount of mutant tissue in the eye increased. On the mutation induction hypothesis this would mean that the supposed mutations would have to take place
at the first larval instar when pigment
potentialities were determined, and that a
decrease in temperature would cause an
increase in mutation frequency (more
mutant tissue). Such suppositions seem
unlikely on several scores. Chen (1948)
and Becker (1960) found that the effective
period for this temperature modification
of pigmentation was during pupation;
temperature shifts in the first larval instar
had no effect. Also, an inverse correlation
between temperature and spontaneous mutation frequency runs counter to the
known positive correlation. Furthermore,
as Becker has pointed out to the author,
the temperature effect cannot be explained
even as an alteration in the time of occurrence of the postulated somatic mutations.
Mutations late in eye development would
produce smaller patches of mutant tissue
than earlier mutations, but the number of
such patches would be greater (more cells
are present to mutate) resulting in the
same total amount of wild-type and mutant areas in the eye. We conclude then
that temperature acts as a modulator of a
predetermined event.
3) No mutations caused by position
effect are produced in the germinal tissue
in variegated flies. If mutations were responsible for the variegation, their occurrence would have to be limited strictly to
somatic tissue.
4) Chromatographic analysis of the pteridines in the eyes of white-variegated flies
indicate amounts of sepia pteridine and
the "Himmelblau" substances in excess of
the amounts found in wild-type heads:
white heads show none of these substances
(Baker and Spofford, 1959; Baker and
Rein, 1962). These excessive amounts are
not observed in flies homozygous for any
of the tested mutant alleles at the white
locus (Hadorn and Mitchell. 1951). This
evidence strongly supports the idea that
variegation is caused by altered gene action
rather than by somatic mutation.
5) The parental genotype—even components of the parental genotype which are
not passed on to the individual being studied—affect the variegation (Spofford, 1959.
1961). It is difficult to conceive how these
could modify the rate of the postulated
GENETIC CONTROL OF DIFFERENTIATION
somatic mutation, but the troublesome alternative remains they they might affect
the timing of a mutational event.
Taken as a whole, these diverse lines of
evidence make it very unlikely that the
mutant areas in variegated tissue are the
result of a change in the basic genetic information in these somatic cells; i.e., to put
it in other terms, any change in the base
sequence of the DNA of these cells. The evidence cited points in the direction of an
alteration in gene action which is inherited
with a fair degree of stability in the
somatic tissue. Therefore, one should consider the processes leading to positioneffect variegation and to cellular differentiation as being analogous: both processes
being the result of somatically inherited
alterations of gene action.
Let us return to a point stressed previously, namely, that the piement-forming
potentialities of the ommatidia in a variegated eye are determined early in development. Tn view of this early determination,
one might not be surprised to find that
pigmentation could be influenced by the
diverse types of genetic information in the
fertilized egg and early embryo that are different from the genetic information of its
own cells. When we first rediscovered (Morgan, Bridges, and Schultz, 1937; especially
Noujdin, 1944) that the parental genotype
influenced the extent of variegation, we had
not recognized that the patterns produced
were indicative of an early determination of
the pigment potentialities. We thought we
were faced with the proposition of having
to find a mechanism for retaining for seven
days (until pigment formation commenced) some of the genetic information
of the parents which did not appear in the
offspring. Happily, as a result of Becker's
work (1957), it is no longer necessary to
postulate that the embryo has such a retentive memory for the parental genotypes.
Of the various types of one-generation
parental effects which have been described
for position-effect variegation (Spofford,
1959, 1961; Baker and Spofford, 1959;
Hessler, 1961; Schneider, 1962), several
may be ascribed to maternal effects. For
example, with the white-variegated system
it has been shown that the type of Y
chromosome in an attached-X mother influences the pigmentation in her variegated
daughters although they do not receive
this chromosome. Another example of
maternal influence in this system concerns
whether the mother is homozygous or
heterozygous for Dp(w+). If the mother
is homozygous, there is more pigment in
the variegated eyes of her heterozygous
offspring than if she were heterozygous.
Similar maternal effects have been observed with peach (an eye color mutation)
variegation evoked by position effect in
another species, D. virilis (Schneider,
1962). There is no compelling reason for
doubting that these maternal effects are
the result of the action of cytoplasmic
materials laid down in the egg by the
mother. Jt is still an open question as to
whether or not these materials act bychanging the time at which the pigment
potentialities are determined during ontogeny.
Aside from parental effects which may
reasonably be ascribed to the maternal
cytoplasm, there is a "parental-source"
effect (Spofford, 1961; Hessler, 1961) that
does not fall so neatly into this category.
Here it is found that with one of the
"states" (these states will be discussed
later) of Dp (w+) there was much more
pigment in the variegated eyes of the offspring if the father rather than the mother
contributed the duplication to the offspring being examined. Representative
data from Hessler are given in Table 1.
The unanticipated observation was that
more pigment was produced when the
mother did not carry the duplication. This
was fascinating, since it was known that
two doses of the duplication in the mother
produced more pigment in heterozygous
offspring than did one dose, and now in
this case the absence of the duplication in
the mother produced about as much pigment as two doses! It is obvious that if a
cytoplasmic maternal effect is involved, it
must be of a complex nature. Perhaps it
might be simpler to suppose that a nucleus
64
WILLIAM K.
BAKER
TABLE 1. The paiental-wurre effect observed in sons. Figures given are units of fluorescence relative to a
standard. (From Hesder, 1961, Genetics.)
Mother's genotype
Father's genotype
Source of
duplication
Drosopterins
Ysru • YL/Y; +/Dp"
\*w • YL/Y; Dp"/+
paternal
maternal
2.95 ±0.17
0.25 ± 0.002
Son's genol)pe
Hessler's data
\hu • Yr'/Y; Dp»/+
Ysw • YL/Y;
y w/Y; +/ +
y w/\'; Dpa/ +
Baker a n d H u b b y ' s data
y in/si* • Y; D p » / +
ywf
Yr- • Ys/scs • Y; H ' / D p "
y w f YL • Ya/sc" • Y; + / D p a
y w f YL • Ys/scs • Y; Bp'/W
paternal
maternal
8.4 ± 1.5
2.0 ± 0.84
V w/scH • Y; D p V H '
ywf
Yr- • Y^fsc* • Y; + / D p »
y <u / YL • Y s /ic» • Y; W/Dp'
ywf YL • Yy.sc 8 • Y; D p * / +
paternal
maternal
33.0 ± 2.9
6.9 ± 2.5
itself has become differentiated by passage
through one parent or the other; i.e., this
parental-source effect is caused by a nuclear differentiation rather than by a cytoplasmic one.
The crosses previously made could not
provide critical evidence on this point
since the mothers and the fathers in the
two crosses were, in fact, of different genetic constitutions. This question has been
resolved by Dr. J. L. Hubby and myself
by measuring chromatographically the red
pigments of the eyes in two types of sons
from one and the same parents. The
crosses used and a sample of the results
are shown in Table 1. The gene W
(Wrinkled wings) is a dominant marker
on the third chromosome which shows
about 1% crossing over with the duplication. In the first cross, the variegated sons
which show Wrinkled wings obtained the
duplication from their mother; whereas,
their brothers that have normal wings received the duplication from their fathers
(males homozygous for the duplication
usually die). The measurements clearly
show that the sons who receive the duplication from their father have more pigment than their brothers whose duplication was of maternal origin. Confirming
results are shown in the second cross in
which the dominant marker Wrinkled is
brought in from the mother. Thus the
parental-source effect is observed with one
and the same parents. Let us reexamine
what this must mean in terms of the information content of the fertilized egg
(refer to Fig. 1). This parental-source effect
must reside in a differentiation of the genetic information in one or the other of
the pronuclei rather than in the extraneous information of the polar body nuclei,
or the cytoplasm since this extraneous information is the same, on the average, in
both types of sons.2 The question then
arises as to whether this is a differentiation
of the female or of the male pronucleus;
i.e., is the pigment forming activity depressed when Dp is passed through the
female or is the activity enhanced through
male passage? There does not appear to
be an experimental way of deciding this
issue. All that can be said is that it must
act on the nucleus bearing Dp rather than
the nucleus bearing w since a deficiency of
the white locus acts as the mutant allelc,
w, in R(w+)/w individuals (Baker, unpublished). Be that as it may, the important point of this work is the demonstration that nuclear components themselves
(but not the white gene) may have been
modified, albeit the nuclear differentiation
lasts only one generation.
8
There is one c)toplasmic difference in the eggs
which give rise to the two types of sons. If Dp has
a paternal origin, then two of the three polar body
nuclei ha\e Dp; whereas, if Dp comes from the
mother, only one of the polar body nuclei contains the duplication. If Dp releases pigmentactivating material through polar body nuclei disintegration, then more of this material would be
in the cytoplasm when the egg has a paternal Dp.
However, the data from the initial crosses in which
this effect was discovered invalidate this argument
since more pigment was produced when the mother
did not ha\e the duplication in her genome.
GENETIC CONTROL OF DIFFERENTIATION
65
Our discussion so far may be summa- netic basis, one state of the duplication
rized by stating that a study of the white- produces variegated eyes with relatively
variegated system has shown that the little pigment and with this state more
pigment potentialities are determined very pigment is produced if the father is the
early in the ontogeny of the eye and that parental source of the duplication. With
these potentialities may be modified, for the other state tested, much more pigment
one generation, by action on the nucleus is produced and there is little if any
containing a gene responsible for pigmen- parental-source effect on pigmentation.
tation, or by direct action of cytoplasmic Cohen studied the following genes that
components in the fertilized egg. As yet affect arrangement of facets in the comwe do not have any experimental infor- pound eye, split, roughest, and facet; (demation on whether the parental-effect noted as "rough eye" in Fig. 5) as well as
modifications act by altering the time of the following genes that affect nicking of
differentiation of pigment potentialities, the wings: facet-notch, faccl-notchoid, and
although this seems possible at least with notchoid. All six of these genes show
cytoplasmic modifiers. If I may be per- position-effect variegation with Dp (tum)
mitted to compound a rash speculation on 264-58a.
top of an unproved assumption, it might
Her findings are interesting. The state
be supposed that the chromosome rear- of the duplication that produced more pigrangement causes the variegated expres- ment in the eye also showed greater areas
sion of nearby loci because of a shift in of normal facet arrangement and a greater
the timing of the time-ordered sequence of number of flies with normal wings (i.e.,
events necessary for normal development. no nicks). Also, if nicks were present, they
A time shift very early in ontogeny might were smaller in size than with the other
well have multiple effects and produce state of Dp. In other words, the expression
alterations in the action of genes whose of the genes acting on characters other
expression is recognized in different organs. than pigmentation was affected in the
Cohen (1962) has obtained some inter- same manner as the white gene. Perhaps
esting information on two questions that of even more significance was her finding
might have a bearing on this notion, (i) that the parental-source effect on the genes
Do the direct effects of the individual's acting on facet arrangement and on wing
own genotype in enhancing or suppressing structure exactly paralleled the parentalpigmentation in white-variegated eyes have source effect on pigmentation.
the same enhancing or suppressing effect
A logical interpretation of these results
on other genes in the duplication, for ex- is that the state of the duplication, as well
ample, genes that determine facet arrange- as the parental-source effect, is acting on
ment or nicks in the border of the wing? the original mechanism producing variega(ii) Does the demonstrated parental-source tion. One is forced to this interpretation
effect of the duplication on pigmentation by the fact that such diverse morphological
operate in the same direction on facet structures as the marginal vein of the wing,
arrangement and on wing nicking?
the arrangement of facets in the compound
The direct effect she studied was the eye, and the pigmentation of the facets are
"state" of the duplication. The genetic all similary affected. The size of the patdifferences responsible for these different terns of disarranged facets in eyes that
states are not yet completely understood, show variegation for roughest or for split
but they must reside either in the duplica- (Cohen, unpublished) indicate an early detion itself, in the heterochromatic region termination of this potentiality, approxisurrounding the duplication, or in some mately at the same time (1st instar larvae)
cases (Spofford, 1962) they are known to as pigment potentialities are determined.
be due to rather closely linked euchro- Now, since parental effects do act early in
matic suppressors. Regardless of the ge- development, and since their action is ap-
WILLIAM K. BAKER
wm258-2l
wm264-58
l'llj. 5 a.—Diagrammatic representation of the results of Schulu (1941) with T(l;4)rum258-2I. The
circles within the eye indicate the areas of "rough
eye" phenotype caused by the split gene, shaded
areas indicate pigment, and white areas represent
no pigment. Note that the white areas are always
rough, b.—A typical variegation pattern with
Dp (wm)264-58a. Note that the white areas may
or may not be rough and that the rough areas
may or may not have pigment.
parently on the original mechanism producing the variegation, then Cohen's results add some credence to the notion that
position-effect variegation might be an expression of a general disturbance in the
sequence of development, perhaps in its
timing.
Since the potentialities for facet arrangement and for pigmentation are both determined very early in development, and
since they both affect the same tissue, the
compound eye, one might raise the question as to whether these processes are related. Could one interpret the polarized
spreading effect discovered by Demerec
and Slizynska (1937) and Schultz (1941)
in this manner? In translocations between
the X and chromosome 4 where the break
in X was either to the right of split or
roughest and either of these genes as well
as the white locus produced a variegated
expression, these investigators found that
in the areas of the eye that were white, the
facets were always disarranged; whereas,
disarranged areas could either have pigment or not have pigment. This situation
is illustrated in Fig. 5a. This was interpreted as a spreading of the suppressing
action of the heterochromatic regions of
the 4th chromosome linearly along the
chromosome, first to either the roughest or
split locus and then to the white locus.
However, one might have interpreted this
interesting finding on the basis of prior
determination of the pigment potentialities placing a restriction on the facetarrangement possibilities. Cohen has
shown (Fig. 5b) that when this region of
the X chromosome is inserted into heterochromatin (i.e., the white-roughest-split
region has heterochromatin on both sides)
then there is no polarized suppression of
gene activity—pigmented areas had disarranged facets and nonpigmented areas
have normally arranged facets. Therefore,
the pigment-forming capacities do not restrict the facet-arrangement potentialities;
these processes are probably independent
of one another. Suppression of gene action
can proceed from either point where the
inserted euchromatin is juxtaposed to
heterochromatic regions.
I should like to close this discussion by
taking another look at the main conclusion
of our investigations, namely, that cellular
differentiation of function consists of a
rather precisely timed alteration of gene action during early ontogeny, an alteration
inherited in the descendant somatic cells.
(I should hasten to add, parenthetically,
that we have no further experimental data
pertaining to this matter, but it may be
instructive to outline our current speculations.) Now, the prime question is the
real meaning of the phrase, "an inherited
alteration in gene action." To state the
question a little more precisely, what are
the mechanisms whereby an inherited
change in gene function can take place
without any change in the elementary genetic information contained within the
gene?
Mechanisms for this type of change have
been studied more precisely in bacteria
than in higher organisms, and perhaps it
might be worthwhile to see what we might
learn by analogy. We have seen that
position effect in Drosophila alters the
gene action of a group of closely linked
loci, sometimes in a polarized fashion. An
GENETIC CONTROL OF DIFFERENTIATION
analogous situation whereby a group of
different genes functions as a unit has been
discovered in Escherichia coli and extensively studied by Jacob and Monod (1961).
Such functional units have been called
"operons." In E. coli they usually consist
of two parts: (i) a series of closely linked
structural genes concerned with enzymatic
control of a single metabolic pathway, and
(ii) a closely linked locus, the operator,
that controls as a unit the activity of these
structural genes. In some cases the operator may effect its control in a polarized
manner, the structural genes closest to the
operator being affected first. It is thought
that in bacteria this control is exercised
by blocking of the formation or transmission of the structural messenger (mRNA)
which carries the genetic information to
the ribosomes where protein synthesis takes
place.
Although the concept of "operons" may
apply to position-effect variegation in
Drosophila, there are three features which
are somewhat different from the analogous situation in bacteria. In the first
place, the genes whose actions are affected
in a polarized fashion are undoubtedly
concerned with quite different metabolic
pathways. For example, we have seen that
eye pigmentation, ommatidia arrangement,
and formation of the marginal vein of the
wing may be affected as a unit. Secondly,
the genes whose actions are altered by
position effect may not be as closely linked
as those genes within an operon of E. coli,
although it is difficult to compare recombinational units in the two forms and relate this to physical distance. Thirdly, the
genes in Drosophila appear to act as an
operon only when there has been disruption of a heterochromatic region (through
chromosome rearrangement) and these
genes have subsequently become attached
to this disrupted heterochromatin.
Let us return now to the question of
possible mechanisms whereby an inherited
change in gene action could take place
and see what one could dream up on the
basis of current concepts about the regulation of protein synthesis. If the basic in-
67
formation of the DNA in variegated somatic cells is not altered, then one could
picture three possibilities: (i) The translation of the DNA information to the messenger (mRNA) is blocked, (ii) The transmission or attachment of the mRNA to
the ribosome is hindered. Or, (iii) the
synthesis on the ribosomes of the proteins
designated by the messengers is blocked.
The second possibility offers some interesting speculations if one considers that
the disrupted heterochromatin may act as
an operator. One could, for example, imagine that one strand of the DNA of
heterochromatic regions of the chromosome is concerned with specifying the high
molecular weight RNA of the ribosomes
and that the complementary DNA strand
designates an RNA that is attached to the
messenger RNA of the linked structural
genes and acts as an operator. (Evidence
that DNA contains a localized sequence
complementary to ribosomal RNA has recently been presented by Yankofsky and
Spiegelman, 1962.) The operon would
then consist of the mRNA of the structural genes located in the euchromatin and
the RNA specified by the adjacent heterochromatin. It seems necessary to assume,
because of the polarized spreading effect
of gene suppression, that the linear continuity of the DNA information in the
chromosome would be maintained in the
messenger. Now, the complementary nature of the purine and pyrimidine bases
in part of the ribosomal RNA and in the
RNA of the operator allows one end of
the mRNA to be bound, through hydrogen bonds, to the ribosome. When the
heterochromatin is disrupted through a
chromosomal rearrangement, an operator
RNA is produced that does not always
bind the messenger to the ribosomal RNA
and therefore the protein syntheses directed by the structural genes do not always proceed.
Now the scheme must fulfill the further
requirement of a relatively stable somatic
inheritance. In other words, if the messenger attaches to the ribosome in a cell
which is at the stage in ontogeny when
68
WILLIAM K. BAKER
the potentialities of gene action are determined, then protein synthesis directed by
the structural genes proceeds normally in
all descendant cells. However, if the attachment is not made, then the descendant
cells will have a mutant phenotype. What
type of self-sustaining mechanism can one
propose to retain in the descendant cells
the knowledge of this prior determination
event? It seems reasonable to postulate
that there are produced at this developmental stage replicating cytoplasmic particles which incorporate the proteins
specified by the structural genes. These
particles would be cell organelles on which
the biosynthesis directed by these proteins
takes place. Only if the entire messenger
is attached to the ribosome, will all the
relevant proteins be incorporated in the
particles. Such a postulate would account
for the polarized spreading effect (if a
given gene has its action affected in part
of the tissue, the genes between it and the
heterochromatic break point are likewise
affected within this tissue spot). In our
imaginary scheme this would mean that
the mRNA of the gene and the remainder
of the messenger between it and the operator would be unattached to the ribosome
and thus none of their proteins synthesized. The self-replicating cytoplasmic
particles would therefore not contain the
proteins specified by these affected genes.3
3
Dr. Throckmorton has pointed out to the author that it seems reasonable to postulate such
particles in view of his (Throckmorton, 1962)
studies on the phylogenetic changes in pteridine
metabolism in Drosophila. He found that most of
the evolutionary steps in pteridine metabolism in
this genus were not changes in the biosynthetic
pathway itself, but were changes in the organ specificity to carry out the reactions. Thus the compound eyes of most Drosophila species synthesize
the drosopterins and sepia pteridine, but only a
few species synthesize both of these compounds in
the testis. In the testis of other species only sepia
pteridine is synthesized, and in the testis of still
others neither compound is made although in both
cases the pigments are produced in the eye. Therefore, the evolutionary changes observed may have
been in the organ specific structure of these postulated particles that carry the machinery for biosynthesis rather than in the chemical reactions per
se leading to pigment formation.
In the hypothesis just presented, the
mosaic gene action in variegated tissue
would not be caused by any alteration in
the structural genes themselves, but rather
by an altered part of the genetic material
that functions in the translation of the
genetic message into specific protein synthesis. As you recall, the experimental
evidence dictates that this is a prime requirement of any hypothesis to explain
position-effect variegation.
Well, it is nice to dream, and I am sure
that each of you could devise just as plausible a scheme. However these nighttime
fancies have a way of disappearing at
dawn when it is realized that their half-life
depends on whether their validity can be
tested. There does not appear to be a
feasible way of testing the scheme just
presented. But contemporary developmental biologists must incorporate into their
thinking the beautifully complex systems
that a cell has for regulating its protein
synthesis. It is the exceptional, and thus
intriguing, behavior of cells, as in variegation, that allows us a glimpse into these
control mechanisms in higher organisms.
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