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The effects produced by centrifuging eggs before and during development.

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THE EFFECTS PRODU(’ED BY C E S T R I F U G I N G EGGS
BEFORE ,4YD DURING DEVELOPMENT.
BY
THOMAS H. BIORGAS.
Columbia linicer8itU.
In recent years the results of experimental embrxology hare led
to the view that however significant in the physiology of the cell,
and even in heredity, the nucleus of the egg may be, the protoplasm
plays also a very important role in early development. I t has long
heen taken for granted by embryologists that the pyesence of such
inclusions as the yolk is an important factor in determining the
fate of embryonic cells. The presence of pigment in certain parts
of the egg, as in the red ring in Toxopneustes, and the yellow band
in Cynthia, has made it possible to follow movements that take
place in the cytoplasm, and these movements are definitely associated
with the formation of organs. The pigment allows us to follow
certain movements that might otherwise escape our attention. The
morements show, at least, that there are accumulations of materials
in certain regions of the egg preparatory to the development of organs
in those regions. How far this accumulation means that previously
esisting differential materials are segregated, and how far it means
that differences are arising in the egg materials, is the point that I
shall here consider.
We owe to E. P. Lyon the introduction of a new method into
enibr-ology by means of which we shift at will rnany of the substances contained in the egg, and in consequence analyze further
their function in embryonic development. The experiments have
already yielded most important results, and I venture to prophesy
that we are only at the beginning of further discoveries.
I f the visible substances of the protoplasm-inclusions I may call
them-act as the initiators of later differentiation, we might expect,
if their distribution is altered, to find an extremely abnormal product.
(155)
156
Thomas H. SIorgan.
I f , on the other hand, the inclusions are not organ-foriiiing materials, we should expect to find iiornial dewlopnieiit after their displaccnient. These alternatives put in sharp contrast the extren1r.s
of expectation. As I shall l’oint out latclr, they do not exclude other
less radical interpretations.
I f the egg, either beforc or after frrtilizatioii, is placed i n the
centrifuge and rotatcd at the rate of aboiit SO00 revolutions pcr
rninutc, a stratification of the niaterials takes place. Thc heaviest
materials are driven to one end, and the lightest to the other. Even
tho niicleiis will be carried through the protoplasm and pass to one
pole of the egg. I n most cases, it appears, that the c g s fall without
regard to their polarity, hcnce tho niicleus aiid the inclusions of
the protoplasm may come to ocwipy positions with respect to thc 1111inovcd parts of the protoplasni that are ciitirelg different from their
normal relations.
I f thc egg is rotated iiiore slowl,~but for a longer time, the same
result is accomplished, a i d thiq method has in somc cases a clccided
advantage. For instance, the egg imincdiatcly after its fcrtilization may be placcd in tho itlachine and kept rotating diiring its early
dcvelopmcnt. I n this way me can iwure against the possilh rvdistribution of thc stratificgi materials.
I need not point out to yon the wondc~fiildelicacy of the centrifugal force, by iiicans of which we can shift at will the contents
of the egg without injuring the egg, as the sequel will show. The
mcthod, as I have said, opens a new era in experimental cytology.
I wish now to bring before 7011 somc of the results already obtained, and, if I speak more especially of thosr forms that I have
studied, i t is Iwctiiise T have here a knowlcdge of the facts at first
hand.
The egg of tho sea urchin Brhacia has given, I think, the most
definite, positive resiilts. Tho egg after centrifiiging is shown in
the first figures of thc diagram (Fig. 1). Fonr distinct ZOIICS are
present--a light whitish cap of oily matter, a mirldle perfectly clear
protoplasm, a band or s c p e n t of yolk and a red pigmented base.
The niicleus lics inst beneath the oil cap.
The normal egg has a definite polarity which, as Roreri s l i o ~ ~ ~ e d ,
The EfFccts I'rocliiced bj- Cciitrifuging Eggs.
Xi
staiids iu rcllation to the point of fixation of the egg to the ovariaii
This point is marked later by thc attachment funnel that
wall.
riiiis tliroiigh the jelly-like ontrr niein1)rane. I t serves, therefore, to
determine the original axis of the egg.
The centrifugcd eggs show that the stratificatioil stands in no
constaiit rcIati011 to thc cgg axis (see first fipnre). I n other mords,
tlir tlgq i.; cmtrifugecl as it h a p p ~ n 4to fall in thr tube.
-------::-_:
__->
---------:-=
- _--
--- --______---:=_ ~
~
-'---
_r
I
4
2
5
3
6
Tht: first cleavage begins, almost invariablg, at the white cap
whcre the nucleus lies and cuts throngh the egg, dividing the stratified
niaterials at right angles (Fig. 2). The second plane is at right
angles to the first (Fig. 3), lying ncar the line of separation of yolk
a i d clear zone. and the third at right angles to both (not shown in
figures). The fourth cleavage is a differential clcarage (Figs. 4,
5 , 6 ) . Fonr micromercs appear at oiie pole; the cells of the opposite
heiiiisphere divide cqiially. This cleavage is a critical one, for the
158
Thomas H. Morgan.
micromes show in the normal egg where the digestive tract will develop. The micromeres become the mesenchyme.
I f the position of the micromeres in the centrifuged egg is followed, it will be found that in these eggs also’ thc archenteron
develops at the micromere pole. Furthermore it has been determined
that the micromeres do not develop with respect to the secondary
induced axis, but lie opposite to the attachment funnel. I n other
words, while the first thrce cleavages come in with respect to the
stratification, the fourth or differential cleavage comes in with respect
to the original egg-axis (Figs. 4, 5 , 6 ) .
Around the micromere pole as a center the development of the
cbmbryo takcs place. A perfectly normal pluteus is formed. I n some
of these plutei the red pigment is contained in one region ; in others,
in other regions. Similarly for the yolk.
The results prove triuniphantly that the materials that ccntrifnge in the egg of the sea urchin are not organ-forming.
Let us turn now to another egg, that of the mollusc, Cumingia.
The former egg, that of the sea urchin, has at first a less determinative type than the mollusc; hence the importance of taking up a
different form.
Three substanccs appear after centrifuging, the yolk at one pole,
the pigment at the other, and a clear zone between.
The first cleavage is shown in the following figures. (Figs.
‘i,
8, 9.) It will be noted that the first cleavage plane pays not the
least attention to the distribution of the materials. All of the
yolk may be in the small cell, or the small cell may contain all of
the pigment. This difference of results may be diie to the fact that
in the sea urchin the nucleus is driven to one pole, while in the mollusc’s egg the polar spindle has developed when the egg is laid. The
centrifuge is unable to change the position of the spindle, although
it can change readily the resulting nucleus. I suspect, however, that
the difference is more profound and that in Cumingia the first cleavage plane is a differential one, like the fourth plane in Arbacia. But
note, it is differential not with respect to the inclusions of thc egg
but with respcct to the polarity of the egg.
F o r some reason unknown to me the eggs of Ciimingia do not
The Effects Produced by Centrifuging Eggs.
159
develop well in dishes as far as the bivalve stage, despite the fact
that the cleavage and early development is quite normal. I have
had great difficulty in obtaining embryos, and this applies equally
to normal and to centrifuged eggs. Whenever I have obtained later
stages in the one, however, I have also obtained them in the other,
so that after a prolonged study I can now state that the centrifuged
eggs of Cumingia are also capable of producing normal embryos.
These results show, I think, that the visible material of these
eggs, such as yolk, oil, pigment and perhaps other granules, are not
necessarily organ-forming or even organ-determining.
1 should, however, give yon a wrong impression if I left the
7
8
9
matter here. I n other eggs it has been shown that the centrifuge
acts injuriously. In the fertilized frog’s egg, for example, I found
that if centrifuging were carried on for too long a time, abnormal
development takes place. Just what happens is not clear. That the
disturbance of the materials may interfere with normal movements
in the protoplasm essential for development is quite possible. I
could give examples of such conditions. It may also be possible
that some of the inclusions are necessary for the cells, as food, for
instance. Their removal from the cells might, therefore, act injuriously. It seems almost a foregone conclusion that even if the
young stages of centrifuged eggs are normal, later stages may become
abnormal or fail to be attained, for with all the yolk in the ectoderm
lG0
Thoinas H. Uorgan.
and iionc i n thc digestive trart we should cxpect abnornial nntrition.
Again the nucleus may be carried so f a r from a poaition essential
to it wlicn thc cliffercntial clearagc occurs that it fails to reach
its proper location at the proper tirnc. I could give evidence to show
that displaced nuclei do endeavor to reach the p r o p r place for a
differential cleavage.
All of these conditions, and probably more, must ultitnately receive
careful attention. Failure of a centrifuged egg to develop normally
may be owing to any one of them, but the positive resiilts show, I
think, with all clearness that the visible inclusions in the cytoplasm
of the kinds here referred to are not organ-forming siibstanccs ; their
r ( J k i 11 dcvelopnient is of svcondary importance.
Behind them lie3
an orgaiiixation that is the chief director of the series of ereiits
that characterize development. The risible materitils of the egg
follow and do not lead in the development.
Both in the case of the sea urchin and of the inollusc I have
spoken of certain cleavages being dil-f'ewntial. T o n ilia? fairly ask
for an explanation of rny meaning; whether niiclear or cj-toplasmic ;
and if the latter how differential if not a separation of different
materials.
Here it seems to me is the really vital question that remains to
be settled by further study. The evidence wc hare does not justify
iis, I thiiik, in snpposing such a cleavage to be thc result of special
nnclcar interference; nor (lo I think it due to the segregation of
ahead- caxistiitg preforineci mat~rials. I ani inclincd to adopt the
view that a differeutial cleavage is onc in which a physical condition
is reached that marks a further step i n development. The clivision is
differential not in the sense of separating thiiigs prerioiisly mixed,
h i t it is a creativc phase by which something new is produced by the
bioplasni. I t occurs at the first division in the mollusc, but at thc
fourth i n the sea urchin. The same kind of change may even go on
before any division has occurred. The resnlts here described for
the egg of two species does not, of course, prcvliide the possibility
that in other eggs a change similar to that which occurs at a differential cleavage may occiir in the egg prior oven to the first cleavage
and after the ripening pcriotl. The results that Conklin has obtained
Tlie Efects Protlucecl by C’cbntrifugiig Eggs.
161
in the egg of &4scidian, C‘yithia, fall iiiider this class. I f , as I
suppose, the early differeiitiation of different regions of the bioplasni
i- :I phgsical fact of the living suLstaiiw, such a chaiige might
readil? take place before the first cleavage. The results of isolatiiig
ljla-toiiivrcs show clcfiiiif(b1y that reiiiu\ a1 ( ~ f11;irts of tho egg, after
a difft~rentialchange has talicii l)lwe, seriously affects in many cases
the snlsrbqnent developiiieiit, ttiid if the wntrifuge is capable of
displacing siich difierential areas or of interfcring with their format ion, or of making ditficult the snbqequrut development by filling
tlieiii with iiitliffercnt mnterials, abnorriiali ties may take place.
I t has been sliowii, cslwcially by C‘onkliii, that extensive Inw cments
of niaterials take place Lcforc the first cleavage and also in subseqiwnt cleavages. Such movements sccm to be connected with the
forniatioii of organ-forniiiig rrgioiis. \\’hc&r siich inovenients mean
tlia t diffcrential iiiatcrials dread? developed are moved to their
clcfinitivc locations, or \&ether the moveiiicints are themsclvcs the
cspri’sj”ionof differential changes taking place, or mhcther such tnoveiiirnts are siniply the ontcoiiir of karyokinctic movements, we do not
know positively.
l’tdiaps 1should atld, in ordcr to prercnt misunderstanding. that,
although I d i s p t e the view that the risible substances i n the sea
iirchin’s C ~ Favtcd ii1wii 1)y tlie ccntrifngc arc organ-forminy, T hold
that devcblopment is the outcome of physical changes in the egg.
The materials that charactcrizc the different structures and organs
of the body are the end product of cliangcs that the egg-substance
undergoes in its development. Differentiation is a product of the
:ietivity of the egg, the egg itself before cleavage is not the sum
total of those materials that characterize the organs of the body.
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