/ . Embryol. exp. Morph., Vol. 16, 3, pp. 469-485, December 1966
With 2 plates
Printed in Great Britain
469
Experimental evidence for the
nuclear control of differentiation in Micrasterias
By G. G. SELMAN 1
From the Institute of Animal Genetics, University of Edinburgh
INTRODUCTION
In its normal biradiate form the desmid Micrasterias thomasiana var. notata
resembles a thin circular biconvex lens about 220 ju, in diameter and 45 ja at its
thickest. The centrally placed nucleus lies within an isthmus connecting two
semicircular semicells. The nucleus is haploid with between 34 and 37 chromosomes (Waris, 19506). Each semicell has one polar lobe directly opposite the
isthmus and two flattened wings to either side of the polar lobe. Each wing is
divided into two side-lobes which are themselves subdivided. At the thin flattened
rim of the cell the wings are differentiated into a notched pattern by which the
individual species may be recognized.
The cells divide in darkness at intervals of about 4 days. The generation time
may be 3 days under the best conditions of culture and may be 5 days or even
longer if the culture medium is in need of renewal. Mitosis is followed at
telephase by the separation of the daughter semicells as a septum grows across
the isthmus between the daughter nuclei (Waris, 1950a). After the septum is
complete the new semicells begin to grow. They attain a hemispherical-bulge
stage and the bulge later puckers into a 5-lobe stage (one polar lobe and two sidelobes for each wing). The wing-lobes then subdivide to give a 9-lobe stage. The
lobes divide again and when the semicells approach their full size the cell walls
thicken as cellulose is deposited and the edges of the wings differentiate into the
notched or dentate pattern. Finally the daughter cells no longer adhere. A semicell is fully formed 6 h after the septum is complete.
Anucleate cells of Micrasterias have been produced after centrifugation
(Waris, 1951) and after treatment with ultraviolet light (Kallio, 1959, 1963) and
in these cases no differentiation of the new hemicells took place. Kallio (1949,
1951, 1953 a, b) has induced polyploidy in Micrasterias, using a variety of
experimental techniques including cold shock, but particularly by centrifugation.
Hyperhaploid cells (Waris, 1958; Kallio, 1959) and hypohaploid cells (Kallio,
1959) have also been produced. From a comparative study of the hemicells which
developed in the presence of nuclei of different ploidies, Kallio (1951) was able to
1
Author's address: Institute of Animal Genetics, West Mains Road, Edinburgh 9,
Scotland.
470
G. G. SELMAN
show that with increased nuclear mass the width of the wings increased relatively
more than their length and the degree of dentation was also greater. In addition
to this nuclear effect on the morphology of the cell, Waris (1950 b) and Kallio
(1951, 1960) have shown that the different pattern of symmetry in uniradiate,
biradiate, tri radiate and tetraradiate forms of Micrasterias may be regarded as
determined by a different cytoplasmic framework which can act independently
of the nucleus.
In the present experiments an ultraviolet microbeam has been used to irradiate
small defined areas of cytoplasm without affecting the nucleus, or alternatively
to irradiate the nucleus without affecting the cytoplasm except for the small
region immediately above the nucleus. The flattened shape of Micrasterias makes
it particularly suitable for this type of study. Kallio (1959, 1963) has treated
Micrasterias with ultraviolet light of wavelength 2537 A and has shown that it is
possible thereby to produce development which closely resembles that of cells
made anucleate by centrifugation. In Kallio's experiments either a whole cell
was exposed to ultraviolet light (Kallio, 1959) or one half of a binucleate double
cell was irradiated while the other half was screened (Kallio, 1963). In either case
both cytoplasm and nucleus were irradiated. The microbeam apparatus enables
small and precisely located defects to be induced at the subcellular level and the
present experiments were conducted to extend our understanding of the growth
and differentiation of the new semicells.
MATERIAL AND METHODS
Stocks of Micrasterias were cultured in the MS medium of Waris (1953).
This is a medium of inorganic salts in dilute aqueous solution, pH 6-0, with the
addition of chelated iron. Artificial daylight was provided by a pair of white
20 W fluorescent tubes each about 1 ft from the cultures. Twelve hours of light
were followed by 12 h of darkness. The dark period was arranged to begin at
10 a.m. by the use of a time switch. The ambient temperature was about 18 °C
and must not rise above 20 °C. Stocks were maintained in sterile medium in
conical flasks with cotton plugs. Experimental cultures were kept in solid watchglasses with a glass cover. Sterile culture appears to be unnecessary if fresh
medium is used every few days.
The slide used for treatments with ultraviolet light was constructed as follows:
a no. 2 coverslip, £ in. square, with a circular hole of 1 cm diameter drilled
through it, was mounted on a standard glass slide. A thin strip of agar gel, the
thickness of a no. 1 coverslip, was then placed on the slide within the hole of the
drilled coverslip. Individual Micrasterias were selected with a small hand micropipette and placed on the agar gel in a drop of medium. A quartz coverslip was
used to cover the Micrasterias by being placed over the drilled coverslip. The
material was thus held flat without risk of damage.
The microbeam apparatus incorporates a microscope with a reflecting
Nuclear control of differentiation
471
objective and is identical to that used and described by Hathaway & Selman
(1961). An intense heterochromatic ultraviolet source is used to provide vertical
illumination over a small area of the field of the light microscope which alternatively uses a conventional tungsten filament source when the specimen is
viewed by transmitted light. The centre of the area within a cell to be irradiated
was focused under the cross-hairs of the ocular. The microbeam had previously
been aligned with these cross-hairs and was of known diameter. The dose of
ultraviolet light was proportional to the exposure time and was controlled by a
photographic shutter in the ultraviolet beam.
MICROBEAM EXPERIMENTS ON THE CYTOPLASM
In the first series of experiments the ultraviolet microbeam was directed at the
tips of certain growing lobes of one of the pair of new hemicells. The earliest
stage at which this treatment was given was the puckered-bulge stage when the
lobes first appeared, and the latest stage of treatment was with each hemicell at
the 9-lobe stage. The other new hemicell was always left untreated as a control
and showed normal development in all cases. The diameter of the beam was 20 fi
throughout all experiments, so that a single irradiation covered an area slightly
less than that of a single lobe. A single irradiation of 60 s duration was found just
sufficient to inhibit entirely the further growth of the treated lobe. A dose of 30 s
duration produced a lesser but unmistakable defect. These treatments did not
affect the nucleus, which remained near the isthmus throughout all stages.
In a typical experiment a Micrasterias was selected at the 9-lobe stage, and
treated at the tip of each of the four lobes of one wing with a separate 60 s dose
of ultraviolet radiation (Plate 1, fig. A). The treated lobes stopped growing and
remained as undifferentiated rudiments about two-thirds of their full length, but
the growth and development of all other lobes continued in a normal manner.
When fully developed, the treated Micrasterias therefore had one obviously
defective wing in one hemicell while its other hemicell and the sister Micrasterias
from the division were entirely normal. The pair of Micrasterias were kept in
normal culture conditions and observed at the next division. All the new hemicells
were normal (Text-fig. 1), including that developed from the old hemicell with the
induced defect (Plate 1, fig. B). All new hemicells were normal at the next three
divisions also. This result is perfectly understandable on the assumption that the
undamaged nucleus controls the development of the wing. There is no evidence
here for any cytoplasmic determination by elements whose function might have
been impaired by treatment with ultraviolet light. All fifteen similar experiments
also gave this result and this behaviour is represented diagrammatically in
Text-figure 1.
In an experiment with a Micrasterias selected at a puckered-bulge stage (Plate
2, fig. L), a 60 s single dose of ultraviolet radiation was given to that part of the
expanding surface of one hemicell which corresponded to a lower side-lobe.
472
G. G. SELMAN
When the hemicell was fully developed it lacked half of one wing nearest the
sinus. This half-wing would normally have developed from the lower side-lobe.
After the following divisions the new hemicell was quite normal, while the old
hemicell remained defective (Plate 1, fig. C). Defective hemicells never recover
after their normal period of growth and development. This particular defective
u.v.
First
generation
Second
generation
Old
New New
Old
Old
Old
New New
d'
Text-fig. 1. A diagram to illustrate a defective wing which formed in a Micrasterias
after treatment of the tips of growing wing-lobes with ultraviolet light. After the next
division all new hemicells are shown to have developed normally.
hemicell was photographed in the 10th generation after treatment (Plate 1,
fig. D) and was shown to have identical form to that which it possessed in the
second generation after treatment. This Micrasterias with the defective hemicell
was subsequently observed in the 14th generation and was then lost by accident.
Individual hemicells when labelled by defect can thus be made to give information about longevity.
In another experiment a single dose of 5 min ultraviolet irradiation was applied
at the 9-lobe stage with the beam across both parts of a lower side-lobe. This dose
was ten times greater than that required to produce a clearly observable localized
defect. In this case not only was the lower side-lobe absent but all the lobes of the
treated hemicell were stunted and without differentiation. A general deleterious
effect has been superimposed on the localized defect. During a 5 min irradiation, the normal streaming movements of the cell would be expected to carry
any deleterious products of irradiated cytoplasm throughout the growing semi-
Nuclear control of differentiation
473
cell. A clouding of the cytoplasm was observed by light microscopy immediately
after the treatment. Nevertheless, even after this drastic treatment and result,
the subsequent division took place after a normal period, and the new hemicell
was almost but not quite normal (Plate 1, fig. E), the corresponding lower sidelobe being defective although it was as long as the normal lobes. At the next
division a normal hemicell was derived from the slightly impaired hemicell
(Plate 1, fig. F), while a slightly impaired hemicell resulted from the originally
stunted abnormal hemicell (Plate 1,fig.G). At subsequent division this behaviour
was similar but by the eighth division and later the new hemicell derived from the
originally stunted hemicell was normal although slightly smaller than in control
material (Plate 1,fig.H). By the eighth generation, therefore, the stunted hemicell
had fully recovered its ability to give rise to hemicells of normal form at each
successive division. The results of this experiment indicate that the nucleus had
not been damaged by the treatment which severely affected the structure 'of one
entire hemicell. There is also evidence in favour of the damage and subsequent
recovery of a cytoplasmic determinant for the lower side-lobe. On the other
hand it perhaps should be remembered that under stress situations (such as
adverse conditions of culture, which do not apply here) the lower side-lobes are
often poorly developed.
A Micrasterias in the interphase between divisions was given four doses each
of 30 s duration to the outer edge of one mature wing. The chloroplast immediately retracted from the treated region. Subsequently the chloroplast appeared
very ragged and abnormal, but after 7 days, which was a longer interphase period
than normal, the Micrasterias divided to give normal cells.
In a fully developed Micrasterias the polar lobes are obviously different in
form from the wing-lobes and they have certain different functions concerned
with cell adhesion. They were also shown to behave differently in the present
experiments. For instance, a single 60 s ultraviolet treatment was applied to the
tip of a polar lobe at the 5-lobe stage, and afterwards the polar lobe was observed to be only about three-quarters of its normal length and of a more rounded
form than is normal (Plate 1, fig. I). Although the untreated sister cell from the
division subsequently divided normally, the Micrasterias with the defective polar
lobe never divided again. It assumed the dark green colour associated with an
increase of chloroplast and appeared fairly healthy for about 24 days, when
degeneration occurred. Another similar experiment with a 30 s dose of ultraviolet light applied to the polar lobe produced a similar result, which is quite
unlike the behaviour of cells with defective wing-lobes, which invariably continue
to divide. Abnormal polar lobes seem to have a more deleterious effect on the
cell than do abnormal wing-lobes. In other experiments very slight polar lobe
defects were induced with slightly lower doses of ultraviolet light and in these
cases subsequent divisions did take place. All the new semicells in these divisions were normal.
474
G. G. SELMAN
OBSERVATIONS WITH DINITROPHENOL
In another series of experiments, abnormal development of hemicells was
induced by the addition of dinitrophenol to the medium. Kallio (1963) used
0-001 M dinitrophenol to retard development so that added ribonuclease might
penetrate Micrasterias to a greater degree. In our case only 0-0002 M dinitrophenol was applied to several Micrasterias for 8 h during the period of lobe
formation. Development ceased in the presence of the dinitrophenol and when
treated cells were returned to a normal medium the lobes remained stunted and
rounded (Plate 1, fig. K). These cells did not die, however, and gave only normal
hemicells in subsequent divisions. In another experiment 0-0002 M dinitrophenol
EXPLANATION OF PLATES
All photographs are of living cells except M and P. The scale represents 50 /i in all cases.
PLATE 1
Fig. A. A normal pair of Micrasterias at the 9-lobe stage. Black circles indicate the position
of four separate doses of ultraviolet radiation, each of 60 s, which were given at that stage.
Fig. B. The abnormal hemicell which resulted from the above treatment (fig. A) has the stunted
wing. The normal hemicell was derived from the abnormal hemicell at the subsequent division
which took place 6 days after the ultraviolet treatment.
Fig. C. The abnormal hemicell resulted from a 60 s dose of ultraviolet light given at the
puckered-bulge stage (fig. L) to the edge of a lower side-lobe while it was forming. The normal
hemicell was derived from the abnormal hemicell at the division after treatment.
Fig. D. The abnormal hemicell is that shown infig.C, but the normal hemicell was derived
from the abnormal hemicell at the 9th division after the ultraviolet treatment. This photograph
was taken from the opposite side of the Micrasterias to that offig.C.
Fig. E. The badly stunted hemicell resulted from a dose of 5 min ultraviolet light given across
a lower side-lobe of an early 9-lobe stage. The more normal hemicell was derived from the
stunted hemicell at the division following the treatment.
Fig. F. The left-hand hemicell is also shown infig.E and has one defective lower side-lobe.
The right-hand hemicell is normal and was the new hemicell derived from its partner two
divisions after the treatment.
Fig. G. The right-hand hemicell is the same stunted one shown infig.F. The left-hand hemicell is more normal but with a defective lower side-lobe. It is the new hemicell derived from the
stunted hemicell two divisions after the treatment.
Fig. H. The stunted right-hand hemicell is that shown infig.E andfig.F. The other hemicell
is normal but small and is the new hemicell derived from the stunted hemicell at the seventh
division after the treatment.
Fig. I. One hemicell has a short and rounded polar lobe. This was the result of a 60 s dose
of ultraviolet light to the tip of a polar lobe at the 5-lobe stage. This cell did not divide
again.
Fig. J. The abnormal hemicell has a stunted polar lobe and deep clefts between the side-lobes
of each wing. The abnormalities developed after treatment with 0-0002 M dinitrophenol for
3 h at the 5-lobe stage.
Fig. K. The abnormal hemicell with stunted lobes resulted from exposure to 00002M dinitrophenol for 8 h at the 9-lobe stage.
/. Embryol. exp. Morph., Vol. 16, Part 3
G. G. SELMAN
PLATE 1
facing p. 474
J. Embryol. exp. Morph., Vol. 16, Part 3
PLATE 2
R
G. G. SELMAN
facing p. 475
Nuclear control of differentiation
475
was applied for only 3 h at the 5-lobe stage, after which the Micrasterias was
washed and allowed to develop in normal medium. Surprisingly this Micrasterias
suffered a stunting of the polar lobe alone while the wing lobes continued to grow
and differentiate (Plate 1, fig. J). There was also an unusually wide gap between
the two halves of each wing lobe. It is clear that dinitrophenol can induce
developmental abnormalities in Micrasterias and the polar and wing lobes may
be affected differently by the treatment.
MICROBEAM EXPERIMENTS ON THE NUCLEUS
In the following series of experiments the nucleus of Micrasterias was irradiated with ultraviolet light but no cytoplasm was irradiated save for the thin
layer immediately above the nucleus. The circular outline of the microbeam,
20 ft in diameter, was made to coincide with the outline of the nucleus viewed
with the microscope. The stage at which the nucleus was treated varied in
PLATE 2
Fig. L. A normal pair of Micrasterias at the puckered-bulge stage. The drawn circle indicates
the position of a nucleus at this stage just before it is obscured by the migration of the chloroplast from the old hemicell. Puckering indicates the position of five lobes seen at the subsequent
developmental stage.
Fig. M. The skeleton or cell-wall outline of a cell whose nucleus was given ultraviolet light
for 30 s at the puckered-bulge stage (fig. L). After treatment, growth continued and the sidelobes of each wing subdivided twice before the cell burst.
Fig. N. One hemicell shows the anuclear type of development, with all lobes rounded and
longer than normal. This development occurred after 5 s of ultraviolet light to the nucleus at
the hemispherical-bulge stage. The abnormal hemicell has stopped growing and normal
thickening of the cell walls has taken place. Subsequently this cell divided to give two normal
new hemicells.
Fig. O. One hemicell shows partial differentiation as a result of a temporary inactivation of
the nucleus which followed from a 2 s dose of ultraviolet light to the nucleus at the hemispherical bulge stage.
Fig. P. The skeleton or cell-wall outline of a uniradiate Micrasterias developed after treatment
with actinomycin D for 160 min at the bulge stage. The polar lobe and divided wing sub-lobes
can be seen but no cell-wall thickening or differentiation took place.
Fig. Q. The abnormal hemicell shows anuclear development after treatment with actinomycin
for 150 min beginning at the puckered-bulge stage. It survived 40 days after treatment and
gave rise to normal hemicells in six divisions.
Fig. R. The abnormal hemicell shows partial differentiation after treatment with actinomycin
for 70 min during early bulge to hemispherical-bulge stages. The development may be interpreted as due to nuclear inactivation followed by nuclear recovery.
Fig. S. The abnormal hemicell with rounded lobes was treated with actinomycin at the 15lobe stage for 60 min. No differentiation has taken place after the treatment.
Fig. T. This Micrasterias developed after treatment with actinomycin for 60 min at the 9-lobe
stage. One hemicell has wing sub-lobes slightly narrower than normal near their tips, where
partial differentiation has taken place after a temporary inactivation of the nucleus.
476
G. G. SELMAN
different experiments from a very-small-bulge stage soon after the septum was
complete to the puckered-bulge stage just before the nucleus was obscured by
the advance of the chloroplast from the old semicell.
In sixteen experiments the nucleus was irradiated with doses of ultraviolet
light for periods of between 6 and 60 s. In each case the hemicell continued to
grow; lobes and sub-lobes formed but the growth period was not followed by a
stage in which thickening of cell walls occurred, nor did differentiation take place.
Growth continued beyond its normal extent so that lobes formed which were
longer than normal. Death followed from rupture of the thin cell walls at a point
near the middle of the hemicell (Plate 2, fig. M). No difference in form was noted
between the results of irradiation for 6 and 60 s, so that irreversible nuclear
inactivation probably resulted from each treatment. Kallio (1963) showed that
Micrasterias thomasiana may form only five lobes after denucleation following
centrifugation or following a lethal dose of ultraviolet light given to an entire
cell in mitosis. Therefore, the ability of lobes to subdivide further may depend
upon the release of determinants by the nucleate cell after the completion of the
sinus. In the present work ultraviolet irradiation of the nucleus gave rise to semicells of the anuclear type with between 7 and 9 lobes, provided the nuclear
inactivation was made less than 15 min before the start of the hemisphericalbulge stage. The hemispherical-bulge stage begins when there is a single point of
contact, rather than a surface of contact, between the daughter semicells, and it is
estimated to begin 120 min after the nuclear mobilization stage (Kallio, 1951) or
45 min after the start of the rapid growth of hemicells. When nuclear inactivations were made at times less than 50 min after the start of the hemisphericalbulge stage, then anuclear development occurred and between 11 and 16 lobes
were formed. In these cases the four wing-lobes formed at the puckered-bulge
stage had divided twice, so that lobe formation followed by two successive
lobe divisions occurred after nuclear inactivation.
In further experiments the nucleus was treated with ultraviolet light at the
hemispherical-bulge stage in each case, but for lesser periods. A 4 or 5 s dose
resulted in development exactly similar to the inactivated-nucleus type described
above, except that these cells did not burst. Lobes were rounded and longer than
normal but thickening of cell walls did finally take place, growth ceased and the
cells survived (Plate 2, fig. N). However, no differentiation took place. After a
normal interval these cells divided and all the new hemicells were normal, as they
were in subsequent divisions. It seems clear that the nucleus in this case had lost
its function only temporarily and was able to recover later. It also follows that
the normal limitation of the growth period and thickening of the cell walls are
controlled by the nucleus.
In another similar experiment the nucleus was treated with ultraviolet light
at the same hemispherical-bulge stage, but in this case for only 2 s. After this
lesser dose the subsequent development, although abnormal, was markedly less
so than after the 4 and 5 s dose described above. In this case some differentiation
Nuclear control of differentiation
All
occurred and gave rise to some of the typical dentate or notched pattern at the
tips of the wing-lobes (Plate 2, fig. O). The polar lobe was still abnormally
rounded at the tip and all lobes were narrower although of similar length to
normal. The nuclear function was assumed to have been interrupted for a short
period as a result of the ultraviolet treatment of the nucleus. In the previous
experiment the nucleus probably did not recover in sufficient time to initiate
differentiation. During a period of nuclear inactivation the sides of a lobe tend
to grow parallel to each other rather than radially. The nuclear inactivation
experiments provide evidence for the initiation by the nucleus of the differentiation process. It was concluded that differentiation must depend upon the presence of a nucleus which is active for some period between the puckered-bulge
stage and the stage at which differentiation is observed. That the time of nuclear
activity responsible for differentiation has not been more precisely determined is
due to the masking effect of the chloroplast on the nucleus. As in the previous
experiment, the abnormal cell divided again after a normal interval and in this
and subsequent divisions the new hemicells were all normal. In a similar experiment, but with ultraviolet irradiation to the nucleus for only 1 s, the subsequent development was entirely normal.
In five experiments ultraviolet irradiations of 15 or 30 s duration were made
at the 9-lobe stage over a position near the isthmus where the nucleus was
thought to be. The nucleus could not be seen with the microscope because of the
presence of the chloroplast. Normal development resulted in each case. The cells
were subsequently observed to give rise to several generations before they were
discarded. There is probably protection of the nucleus by the chloroplast.
Kallio (1963) observed a low sensitivity of the later stages of development to
ultraviolet irradiation. The uniradiate form of Micrasterias thomasiana was
shown to behave similarly to the biradiate form when the nucleus was treated
with ultraviolet light at early developmental stages. The above experiments were
therefore repeated with four uniradiate Micrasterias at the corresponding later
stage, because it was thought that protection of the nucleus by the chloroplast
might be less effective in the uniradiate form. In three cases the subsequent
development was normal and in the fourth case there was a slight general stunting at the tips of the wing-lobes. The treated cells did not divide again although
the control partners divided. One of the cells (not the one which showed slight
stunting) burst immediately it was fully grown. The other three showed a dark
green chloroplast after 3 or 4 days and degenerated after about 10 days. The
optical opacity of the chloroplast has made the performance and interpretation
of these experiments uncertain. If only nuclear damage was suffered by the uniradiate Micrasterias, then the results could be taken as meaning that nuclear
determination of the limitation of cell growth and the onset of differentiation are
complete by the end of the 5-lobe stage which corresponds to the 9-lobe stage of
the normal biradiate form.
When the nucleus of a biradiate Micrasterias in the interphase between
30
JEEM l6
478
G. G. SELMAN
divisions was treated with ultraviolet light for 30 s, either of two results was
obtained. In the first case the period before the next division was unusually
prolonged. A very dark green chloroplast was observed after about 4 days and
normal divisions began after about 10 days. In the second case, no subsequent
divisions took place and degeneration set in after about 20 days.
A few experiments were made with a smaller microbeam of 11 fi diameter
which was directed for 5 s to one side of the nucleus at the hemispherical-bulge
stage of development. The inactivated-nucleus type of development was obtained, followed by cell recovery without differentiation, and in another case
partial differentiation was obtained. The results were identical to those obtained
by treating the whole nucleus with the larger microbeam for periods of between
2 and 5 s, and no asymmetrical development was observed.
OBSERVATIONS WITH ACTINOMYCIN D
When experiments are conducted with an ultraviolet microbeam there is no
doubt about the position of the damaged area within the cell or the time at
which that damage is inflicted. On the other hand, when injury or abnormality is
produced by exposing the cell to a metabolic inhibitor there is usually some
uncertainty about the time required to reach a given concentration at a site within the cell, and any localized damage is dependent upon such specificity as the
inhibitor may possess. Kallio (1963) demonstrated the anucleate type of development in Micrasterias exposed to ribonuclease at 0-05 mg/1. Actinomycin D is
known to inhibit the synthesis of RNA in the nucleus by binding to the DNA
template (Goldberg & Rabinowitz, 1962; Goldberg, Rabinowitz & Reich, 1962;
Reich & Goldberg, 1964) and it has also been shown to suppress differentiation
in a variety of developmental systems when used at appropriate concentrations (Gross & Cousineau, 1963; Denis, 1964; Jainchill, Saxen & Vainio,
1964; Laufer, Nakase & Vanderberg, 1964; Wessells, 1964; Yamada & Roesel,
1964).
In Micrasterias preliminary experiments at low to moderate concentrations of
actinomycin D failed to produce abnormal development. Some further experiments were then made with a high concentration (100 /^g/ml) of actinomycin D
for limited periods after which the Micrasterias were washed in several changes
of normal medium. In the presence of this concentration of actinomycin the
hemicells did not develop for more than 30-40 min beyond the stage at which
they were placed in contact with the inhibitor but development was resumed in
the normal medium.
When the Micrasterias were exposed for more than 2\ h the subsequent development was always of the anuclear type and the cells finally burst. For example,
when a uniradiate Micrasterias was treated for 160 min beginning at a smallbulge stage, it progressed to a large-bulge stage before it was returned to the
normal medium and subsequently grew five lobes which were longer than normal
Nuclear control of differentiation
479
before death (Plate 2, fig. P). The corresponding experiment with a biradiate
cell gave anuclear development and nine lobes were formed.
A Micrasterias was exposed for exactly 150 min beginning at the puckeredbulge stage and when it was returned to normal medium both new semicells
grew with a typical anuclear pattern of development. The lobes grew larger than
is normal (Plate 2, fig. Q) but thickening of the cell walls took place and the
daughter cells disjoined. One was lost accidentally but the other survived to
give normal hemicells in subsequent generations. In other experiments the bulge
stage of development was treated for 80 min before the substitution of normal
medium. In these cases the anuclear pattern of development was again obtained
(although the arms of the lobes were narrower than the example of Plate 2,
fig. Q) but the cells recovered and divided again in 5 days to yield normal new
hemicells in all cases. One 15-lobe stage was treated for 60 min with the inhibitor
and it developed only rounded tips to the lobes with no dentate differentiation
(Plate 2, fig. S) but was otherwise normal, and both daughter cells divided again
normally after 5 days. From this observation it seems legitimate to conclude
that the messenger RNAs which transmit the nuclear control over differentiation
must be synthesized after the 15-lobe stage or immediately before the differentiation is observed to take place. However, this conclusion would only follow if one
could be sure that the actinomycin D in this case only acted by preventing the
formation of DNA-primed RNA synthesis in the nucleus.
Two bulge stages were exposed to actinomycin D at 100 /*g/ml for 60 min before
they were returned to normal medium. In this case one each of the daughter
cells did not survive the treatment but the other recovered and showed partial
differentiation (Plate 2, fig. R) in lobes which were slightly longer but thinner
than normal. These cells were remarkably similar to those which showed nuclear
recovery and partial differentiation after ultraviolet irradiation of the nucleus
(Plate 2, fig. O). These cells treated with actinomycin also divided again after
5 days and gave normal new hemicells. The close similarity in the results obtained
after ultraviolet treatment of the nucleus and after actinomycin supports the
idea that in the latter case, also, the control over differentiation exerted by the
nucleus is being affected.
Another Micrasterias was exposed to the same concentration of actinomycin
for 60 min at the 9-lobe stage. The new hemicells both recovered and showed
partial differentiation at the tips of the lobes (Plate 2, fig. T). In this case only the
tips of the lobes were narrower than normal, presumably because of the later
stage of treatment. After 5 days the next cell division took place and normal new
hemicells were produced.
DISCUSSION
The experimental results with the microbeam have shown that about ten
times the dose of ultraviolet radiation required to inhibit a growing cytoplasmic
lobe is required to inactivate the nucleus. For this reason Kallio (1963) was
30-2 •
480
G. G. SELMAN
able to obtain the anuclear pattern of development after irradiation of a whole
cell, or half a double cell, during division stages. The 'standard lethal dose' used
by Kallio (1963) was sufficient to inactivate the nucleus without producing a
significant inhibition of the growth of the cytoplasmic lobes.
In the present experiments, irradiation of the nucleus with a particular dose of
ultraviolet light was followed by the anuclear type of development and subsequent cell recovery with the ability to undergo normal division. When Kallio
(1963) used ultraviolet light to obtain anucleate-shaped semicells from normal
mononuclear cells, these cells never divided again. On the other hand, when
Kallio (1963) used binucleate double cells and gave each nucleate half a standard
lethal dose separately with a 1 h interval between irradiations, the new double
cell survived.
For the recovery of the Micrasterias nucleus following ultraviolet irradiation
no attempt was made in the present work to assess the possible role of photoreactivation. Visible light was used to make observations on the treated cells
at intervals after treatment with ultraviolet light.
The similarity in the appearance of the abnormal hemicells after ultraviolet
treatment of the nucleus and following exposure to actinomycin D is remarkable.
In both cases cell recovery was possible after an anuclear type of development
and partial differentiation was observed with a lesser dose. The fact that in this
case the concentration of actinomycin required to produce abnormalities is so
much greater than the concentrations of about 0-05 /*g/ml required by mammalian cells, for instance (Jainchill et al. 1964; Wessells, 1964), must cast some
doubt upon the specificity of the inhibitor for primer DNA in the circumstances
of the present work in spite of the fact that the induced abnormalities are all
of the anuclear type. Acs, Reich & Valanju (1963) have suggested that actinomycin D can also depolymerize RNA. Other workers have found it necessary
to employ high concentrations of actinomycin D in developmental studies.
Brachet & Denis (1963), for instance, used concentrations between 10 and
20 ^g/ml over 8-10 days in darkness to produce clear inhibition of morphogenesis in Acetabularia, and McCalla & Allan (1964) used concentrations of
30-45 /Ag/ml for 12 h to inhibit chlorophyll formation in Euglena. To produce
rapid inhibition, as in our case, even higher concentrations may be required.
Paul & Struthers (1963) found that 100/ig/ml were required to stop RNA
synthesis in a certain strain of mouse fibroblast cells, and Gross & Cousineau
(1963) used 20-100 /<g/ml to suppress differentiation while allowing cell division
to continue in sea-urchin embryos.
For all those aspects of development in Micrasterias which depend upon
nuclear determination, it is possible to carry out enucleation or nuclear inactivation experiments at particular developmental stages and, by observing the subsequent development, to draw conclusions concerning the stage at which the
determinants leave the nucleus. Conclusions drawn from individual experiments
have already been stated after the description of the experiment and they are
Nuclear con trol of differen tia tion
481
also collected in Table 1 together with those derived from the work of Kallio.
The time-scale used in the table is reckoned from the moment of nuclear mobilization (Kallio, 1951) or breakdown of the nuclear membrane before mitosis, and
is based on an analysis of a time-lapse film made by Mr E. C. A. Lucey of this
department's Research Film Unit. The conclusions should not all be regarded
Table 1. Determination in Micrasterias development
All times in the table are reckoned from the nuclear mobilization stage
Developmental event
Position of septum
Initiation of septum
Main lobe formation and
symmetry
Division 3 to 5 lobes
Divisions 5 to 9 lobes
Division 9 to 17 lobes
Growth limitation
Notched-wing
differentiation
Time of
nuclear
Time of
determievent
Type of
nation
(min) determination (min)
30
30
160
Cytoplasmic
Nuclear
Cytoplasmic
160
Nuclear
215-230 Nuclear
275-280 Nuclear
Nuclear
320
320
Nuclear
Source of evidence
—
Kallio (1963)
Kallio (1951, 1959, 1963)
Waris (1950)
Kallio (1961)
Waris & Kallio (1964)
45-70 From Kallio (1963)
100-110 Microbeam
120-130 Microbeam
As for
Microbeam
differentiation
—
Enucleation. Waris (1951)
—
Quantitative dependence.
Kallio (1951)
After 165 Microbeam with M. birad.
Microbeam with M. unirad.
Before
0
—
240
After 275 Actinomycin D
as carrying equal weight and most require confirmation from future experimental
studies. In Micrasterias thomasiana anucleate cells often develop 5-lobed
hemicells but may develop only three lobes. When Kallio (1963) produced
anucleate-shaped cells by ultraviolet irradiation of the whole cell at the time of
septum formation, then 3-lobed cells developed after a 6 min dose but 5-lobed
cells after only a 2-3 min dose. This suggests that nuclear control of the splitting
of the wing-lobes operates at this stage, which is about 2 h before the splitting
takes place. Kallio (1963) states that when the cytoplasm of anucleate Micrasterias thomasiana is irradiated, so as to inactivate all the RNAs, only 3-lobed
new semicells are formed. The present work also indicates that nuclear determination of the further subdivision from 5 to 9 lobes takes place just before the
start of the hemispherical-bulge stage and determination of the next subdivision
just after that stage. The number of lobes formed when anuclear development
followed after treatment with actinomycin was consistent with the pattern of
482
G. G. SELMAN
determination derived from the microbeam experiments on the assumption that
nuclear determinations cannot act in the presence of the inhibitor. The present
microbeam experiments on nuclear inactivation with biradiate Micrastehas
show clearly that nuclear determination of growth limitation and differentiation
takes place after the puckered-bulge stage. If one accepts the weaker evidence
from the experiments with uniradiate Micrasterias one should infer that nuclear
determination of differentiation acts at or before the 9-lobe stage in the biradiate
form. On the other hand, if actinomycin D in the present experiments only inhibited the production of new messenger-RNA in the nucleus one should infer
that nuclear determination of differentiation does not occur before the 15-lobe
stage. If activation of previously formed messenger-RNA also took place, then
this conclusion would not follow. In that case any stage of development could be
interrupted or prevented by the inhibitor and no conclusions could be drawn
about nuclear determination. Nevertheless, the broad conclusion may be drawn
that for Micrasterias there is evidence for nuclear determination acting at times
between 30 and 155 min before the particular developmental events are observed
to take place under the light microscope.
In the present experiments with the ultraviolet microbeam directed upon
certain cytoplasmic regions no evidence was obtained for determination by
cytoplasmic axes, save for the case of one lower side-lobe after a heavy dose of
ultraviolet irradiation. The negative evidence should not be taken as casting
doubt on the existence of cytoplasmic axes such as have been postulated to
exist by Waris (1950a, b), Kallio (1951,1960) and Waris & Kallio (1964), as it is
unlikely that the axes are particularly sensitive to ultraviolet irradiation. Since
they would be expected to exert their determination of the basic pattern of lobes
by growth across the isthmus soon after the septum is formed, it would be
difficult to affect their growth with ultraviolet light without damage to the
nucleus.
SUMMARY
1. After individual cytoplasmic lobes of growing hemicells of Micrasterias
were irradiated for 30 or 60 s with a microbeam of ultraviolet light 20 ft in
diameter, the corresponding parts of the fully developed wings were stunted.
In subsequent divisions the new hemicells were normal.
2. In one case a hemicell, grossly malformed as a result of a 5 min dose of
ultraviolet light to the cytoplasm, did not give a completely normal hemicell in
the first seven divisions after treatment, although it did so in subsequent
divisions.
3. Abnormal development was induced with dinitrophenol.
4. The ultraviolet microbeam irreversibly inactivated the nucleus when
applied to it for longer than 5 s. Growth continued after nuclear inactivation but
neither thickening of the cell walls nor differentiation took place.
5. After applying the microbeam to the nucleus for 3-4 s, an anuclear type
Nuclear control of differentiation
483
of development was followed by cell-wall thickening without differentiation.
Subsequent divisions took place normally.
6. After applying the microbeam to the nucleus for 2 s, partial differentiation
took place and subsequent divisions were normal.
7. By allowing hemicells to develop in the presence of actinomycin D at high
concentrations for short periods, exactly the same patterns of abnormal development followed by cell recovery were observed as after ultraviolet irradiation of
the nucleus.
8. From the experiments, estimates were made of the times of nuclear determinations of lobe-splitting, cell-wall thickening and differentiation. Nuclear
determination appeared to take place between 30 and 155 min before the developmental event was observed under the light microscope.
RESUME
Preuves experimentales du controle nucleaire de la differentiation chez Micrasterias
1. Apres irradiation individuelle de lobes cytoplasmiques d'hemicellules en
croissance de Micrasterias, a l'aide d'un dard ultraviolet d'un diametre de 20 fi
pendant 30 ou 60 secondes, les regions correspondantes des expansions alaires
pleinement developpees etaient atrophiees. Au cours des divisions suivantes,
les nouvelles hemicellules etaient normales.
2. Dans un seul cas, une hemicellule tres malformee, apres une irradiation
ultraviolette du cytoplasme pendant 5 minutes, ne donna pas naissance a une
hemicellule completement normale au cours des sept premieres divisions
suivant le traitement, bien qu'elle l'ait fait au cours des divisions suivantes.
3. Le dinitrophenol a induit un developpement anormal.
4. Le dard ultraviolet a inactive le noyau irreversiblement quand il lui a ete
applique pendant plus de 5 secondes. La croissance s'est poursuivie apres
l'irradiation nucleaire mais ni l'epaississement des parois cellulaires ni la differenciation n'ont eu lieu.
5. Apres application du dard au noyau pendant 3 a 4 secondes un type
anucleaire de developpement a ete suivi de l'epaississement de la paroi cellulaire
sans differenciation. Les divisions suivantes ont eu lieu normalement.
6. Apres application du dard au noyau pendant deux secondes, une differenciation partielle a eu lieu et les divisions suivantes etaient normales.
7. En faisant se developper les hemicellules en presence d'actinomycine D a
forte concentration pendant de courtes periodes, on a observe exactement les
memes types d'anomalies du developpement, suivies d'une restauration cellulaire,
qu'apres irradiation ultraviolette du noyau.
8. A partir de ces experiences, on a estime la duree de la determination
nucleaire pour la fissuration des lobes, l'epaississement de la paroi cellulaire et
la differenciation. La determination nucleaire parait avoir lieu entre 30 et
155 minutes avant que le phenomene soit observe au microscope.
484
G. G. SELMAN
I wish to thank Professor C. H. Waddington for the suggestion that Micrasterias might be a
useful material for the study of nuclear and cytoplasmic determination after making cytoplasmic defects with an ultraviolet microbeam, and for his continued interest during the course
of this work. I am also grateful to Professor P. Kallio for the gift of stocks of Micrasterias and
Messrs Merke, Sharpe and Dohme for the gift of actinomycin D used in these experiments.
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