/ . Embryol. exp. Morph. Vol. 24, 3, pp. 555-582, 1970
555
Printed in Great Britain
Studies on mitochondria in the early development
of the slug Avion ater rufus L.
By A. H. SATHANANTHAN 1
From the Department of Zoology, University of Ceylon
SUMMARY
The structure, distribution, cytochemical nature and functional significance of mitochondria have been studied during the embryogenesis of the slug An'on, from the ovum to
the postgastrula stage.
Mitochondria seem to undergo progressive but profound changes in form and fine structure
during early development and their distribution conforms to a gradient pattern, which more
or less resembles that of the sea urchin.
The most significant event in cyto-differentiation is the appearance of an extraordinary
vegetal aggregation of mitochondria at the 8-cell stage which heralds a vegetalizing influence
and undoubtedly has a precise morphogenetic function. This vegetal aggregation becomes
involved in a remarkable series of morphogenetic movements and its mitochondria are later
incorporated into the cells that differentiate into mesoderm and endoderm. Thus, apart from
their energy-linked functions, mitochondria seem to play a dramatic directive role in development; mitochondrial segregation, in particular, seems to be the important factor in differentiation.
Certain aspects of their distribution and enzymic composition are discussed in relation to
their normal metabolic functions.
The significance of mitochondrial RNA and DNA with reference to protein synthesis and
self-duplication of mitochondria, and the possible roles played by mitochondria in inductive
phenomena are also discussed in the light of recent work.
INTRODUCTION
Recent research has demonstrated unequivocally that mitochondria are the
most vital organelles of the cell (Green, 1964; Lehninger, 1964). Apart from
producing energy and metabolites for all the life processes of the cell, it is now
becoming increasingly apparent that they may have other specialized functions
to perform.
There is considerable evidence to show that mitochondria may play a morphogenetic role in development (Brachet, 1960; Novikoff, 1961; Gustafson,
1965). Their fine structure is also known to change during embryogenesis
(Weber, 1962; Yamamoto, 1964; Berg & Long, 1964).
This investigation deals with certain aspects of mitochondrial structure,
distribution, cytochemical composition and functional significance with relation
to embryonic development.
1
Author's address: Department of Zoology, University of Ceylon, Thurstan Road,
Colombo 3, Ceylon.
556
A. H. SATHANANTHAN
MATERIALS AND METHODS
The slugs were obtained from Aberystwyth, Wales, and were bred outside
the Zoology Laboratory, University of Reading. The eggs were gently decapsulated in tap-water using a binocular microscope and the embryos were rinsed in
isotonic saline (0-28 % NaCl) and examined by various methods. Mitochondria
were studied in ova and in a closely knit series of early embryos up to the
postgastrula stage. The following methods were used: (a) classical cytological
techniques; (b) phase-contrast microscopy and vital staining; (c) cytochemical
methods including enzyme tests on living embryos; (d) electron microscopy.
Centrifuged embryos were also examined by the first three methods. These
were obtained by subjecting the entire capsules to a centrifugal force of 950 g
for 6 min and then decapsulating them immediately afterwards.
(a) Classical methods
Recent modifications of traditional mitochondrial techniques were used
successfully. Embryos were fixed in various fluids like Flemming, Altmann,
Champy and Helly and then postchromed in 3 % potassium dichromate for
3-5 days in most cases. After fixation the embryos were washed overnight and
quickly dehydrated, cleared and stored in 1 % celloidin in methyl benzoate
according to Peterfi's method (Pantin, 1960). Each embryo was then rapidly
embedded in 56 °C ceresin-wax and serial sections (4/* thick) were cut with a
Cambridge rocking microtome. Acid fuchsin was widely used for staining
mitochondria. Altmann's method modified by Metzner & Krause (1928) and
Kull's method after Baker (1950) were used. The best results were obtained by
the Helly-Metzner combination. Heidenhain's haematoxylin used in routine
work in conjunction with Champy, Helly, Perenyi and Ciaccio was also very
useful in the study of mitochondria. Careful differentiation with picric acid,
instead of iron alum, produced good results. With acid fixatives, however,
mitochondrial form was not so well preserved.
(b) Phase-contrast microscopy
Embryos were squashed in isotonic saline and examined under oil immersion
with a Wild M 20 phase-microscope.
Vital staining
This was carried out according to Baker (1958). Embryos were stained in
Janus green B, Grubler's neutral red, Nile blue, toluidine blue and methylene
blue for 5-40 min using a 001 % solution of the stain in isotonic saline. These
were then rinsed and squashed in saline and examined with an apochromatic
oil-immersion objective.
Mitochondria in slug development
557
(c) Cytochemical methods
A variety of cytochemical tests were carried out on both living and fixed
embryos (Table 1).
Enzyme tests
For cytochrome oxidase whole embryos, decapsulated without injury and
with polar bodies intact, were incubated vitally for 15-45 min in Burstone's
medium (Pearse, 1961), and then washed, compressed and examined in saline,
under apochromatic oil-immersion, just as vitally stained preparations are
studied—'vital enzymology'. This vital method proved to be excellent for the
study of cytoplasmic inclusions, not only for this enzyme, but for succinic
dehydrogenase and other enzymes as well. Localization is very sharp and well
defined and the inclusions could be differentiated easily, as in vitally stained
preparations, for Brownian movement is still evident. Further the results are
highly reproducible. Loss of enzyme activity and artifacts due to fixation and
diffusion are virtually non-existent.
For succinic dehydrogenase, Nachlas's nitro-BT method (Pearse, 1961) and
Seligman and Rutenbuig's neotetrazolium method (Gomori, 1952) were used.
Embryos were incubated for periods of £-l£ h (optimum time, 1£ h) to avoid
diffusion artifacts and examined as for cytochrome oxidase. It was found
necessary to use accelerators (Ca 2+ and Al 3 + salts) to obtain quicker results.
For other cytochemical tests appropriate fixatives were used and serial
sections (4/t thick) were obtained using the same technique employed in the
classical methods. All colour reactions were visualized with a Zeiss apochromatic oil-immersion objective.
Illustrations were drawn with a Zeiss camera-lucida and photomicrographs
were taken with a Zeiss photo-microscope using Ilford Pan-F film.
(d) Electron microscopy
Embryos were fixed in plain aqueous 1 % OsO4 for 1 h at about 2 °C, and
then for •} h at room temperature (Baker, 1965). These were then embedded in
Araldite and sectioned with a Huxley ultramicrotome. Sections were mounted
on carbon- and formvar-coated copper grids, stained with uranyl acetate and
examined with an Akashi TRS-50 electron microscope.
RESULTS
(a) Classical methods
Mitochondria stain bright red with Metzner's method while the rest of the
cytoplasm is a transparent pale yellow. With Kull's method the mitochondria
stain crimson red, the ground cytoplasm pale yellow, but the yolk has a bluish
tinge. Albumen vesicles are pale yellow in both cases. Larger, refractile Golgi
558
A. H. SATHANANTHAN
SCM
SCM
ABBREVIATIONS ON FIGURES
AV
C
Ect.
End.
ER
G
GC
HZ
L
LG
M
albumen vesicles
cortical region
ectoderm
endoderm
ergastoplasm
Golgi
granular cytoplasm
hyaloplasm zone
Iysosome
lipid globules
mitochondria
Mes.
OC
P
PM
SCM
SG
VM
ViM
Y
YZ
mesoderm
oil cap
pseudopodium
plasma membrane
subcortical mitochondria
shell gland
vegetal mitochondria
vitelline membrane
yolk granule
yolky zone
Mitochondria in slug development
559
bodies, nucleoli and chromosomes are also stained very intensely while asters
stand out very clearly.
In general, the mitochondria appear as minute, distinct, rounded bodies,
apparently of uniform size, about 0-5/A in diameter. They are found distributed
in the cytoplasm forming circles around almost colourless yolk granules, or are
seen in the interstices between the yolk and other inclusions. They are sometimes seen in small groups or more rarely arranged in linear rows or chains. In
Limnaea, Bretschneider & Raven (1951) describe the formation of small alphagranules (0-5/6 in diameter) from more elongate, filiform mitochondria during
oogenesis. These perhaps correspond to the rounded mitochondria seen in
Arion.
ViM
SCM
Mes.
End.
FIGURES
1-8
Illustrations showing distribution of mitochondria in early embryos.
Fig. 1. Sagittal (Sag.) v.s. mature ovum.
Fig. 2. Sag. v.s. 2-celI stage.
Fig. 3. Sag. v.s. 4-cell stage.
Fig. 4. Sag. v.s. 8-cell stage.
Fig. 5. Sag. v.s. meso-blastula.
Fig. 6. Sag. L.S. gastrula.
Fig. 7. Sag. L.S. postgastrula, shell-gland region.
Fig. 8. Sector of mature ovum showing centripetal gradient distribution of mitochondria.
In densely populated areas mitochondria are very closely packed and seem to
touch one another. A few appear to be somewhat larger. There are more
mitochondria around nuclei situated very close to the nuclear membrane. In the
region of the mitotic apparatus, however, there are comparatively fewer
560
A. H. SATHANANTHAN
11
FIGURES
9-23
Photomicrographs of early embryos, showing distribution of mitochondria, which
appear as fine darkly stained granules.
Fig. 9. Sag. v.s. mature ovum (Altmann/Metzner).
Fig. 10. Sag. v.s. centrifuged ovum at second maturation (Helly/Metzner). Note
stratification of mitochondria in upper hyaloplasm zone.
Fig. .11. Sag. v.s. 2-cell stage (Altmann/Metzner).
Fig. 12. Sag. v.s. 4-cell stage (Helly/Metzner).
Mitochondria in slug development
561
17
Fig. 13. Para-sag, v.s. of ovum at second maturation (Helly/Metzner). Note centripetal gradient distribution of mitochondria.
Fig. 14. Animal region of ovum (above) at higher magnification.
Fig. 15. Vegetal region of ovum (above) at higher magnification.
Fig. 16. Lateral aspect of ovum (above) at higher magnification.
Fig. 17. Sag. v.s. 8-cell stage (Helly/Metzner).
562
A. H. SATHANANTHAN
50//
Fig. 18. Sag. v.s. 8-cell stage (Champy/Metzner).
Fig. 19. Vegetal region of 8-cell stage (above) at higher magnification.
Fig. 20. Sag. v.s. meso-blastula (Perenyi/Heidenhain's haematoxylin).
Fig. 21. Sag. L.S. gastrula (Champy/Kull).
Mitochondria in slug development
563
mitochondria (Fig. 12). The spindles are almost devoid of mitochondria and
very few are seen between astral rays. Mitochondria are arranged to form a
distinct ring around the centrioles. All these observations have been confirmed
in electron micrographs. Soon after each division a finely granular zone of
cytoplasm appears around each newly formed nucleus (Figs. 2, 11). The fine
granules are RNA-positive and are probably microsomes. Mitochondria are
evenly distributed among these granules but they are more numerous towards
the periphery of these masses. The polar bodies too have their due share of
mitochondria which have presumably found their way there during the maturation divisions. This may also explain why the tip of the animal pole (AP), soon
after the maturation divisions, is devoid of mitochondria.
Distribution
The pattern of distribution of mitochondria varies somewhat in different
stages of development.
Ova. Mitochondria are more or less evenly distributed except in the nuclear
region at the AP and in the cortical zone (Figs. 1, 9, 13). The most striking
feature is a thin densely packed zone of mitochondria just inside an agranular
cortical region within the plasma membrane (Figs. 8, 14-16). Mitochondria then
show a gradual decrease in number towards the centre of the ovum. Under high
magnification, starting from the periphery, one sees the vitelline and plasma
membranes, which may be closely apposed to one another, and then a clear
cortical zone (about 1-2/J, in thickness) devoid of any granular inclusions. Just
beneath this 'cortex' is the dense zone of mitochondria (3-4 layers thick) and
then the general cytoplasm with fewer mitochondria, yolk and other granules.
In the general cytoplasm there is a very gradual decrease in the number of
mitochondria as the centre is reached. Very exceptionally one or two yolk
granules may be found within the subcortical mitochondrial zone.
Thus there is a fairly well defined but subtle gradient distribution of mitochondria of a centripetal nature, showing a more or less concentric arrangement
of these granules in the cytoplasm. One has to look at perfectly flattened sections
without asters and nuclei to appreciate this gradient fully. Complete series of
sections of six ova at various stages of development, from second maturation
division to very early cleavage, have been studied and in all cases this gradient
is evident. In later ova, however, there are slightly fewer mitochondria in the
subcortical zone. Their earlier distribution suggests that mitochondria originate
in the subcortical region and later migrate to other regions as development
proceeds. In the cleaving ovum the mitochondria show the same arrangement
around asters as described before.
2-cell stage. There seems to be hardly any difference between this stage and
the late ovum stage. The same gradient is still evident even though there are
inter-blastomeric cell membranes. Mitochondria do not crowd on either side of
the latter as in the subcortical zone so that the general distribution from the
564
A. H. SATHANANTHAN
periphery to the centre of the embryo is very much the same as before. There is,
however, a slight increase in the number of mitochondria in the subcortical zone
of the vegetal halves of the two blastomeres (Figs. 2, 11).
4-cell stage. An early 4-cell stage is very similar to a 2-cell stage. But as the
third cleavage is approached there is a marked crowding of mitochondria in
the subcortical zone at the vegetal pole (Fig. 3). As the third cleavage asters
appear the crowding becomes more and more apparent in the vegetal parts of
the four blastomeres (Fig. 12).
8-cell stage. This is the crucial stage in the early development of Arion. Its
most striking and obvious feature is an intensely staining, densely packed,
pyramid-shaped aggregation of mitochondria at the vegetal pole (VP) occupying
about a fourth of the volume of the megameres (Figs. 4, 17-19). This enormous
massing of mitochondria is the most significant event in the process of cytodifferentiation in Arion and has profoundly affected the general pattern of
distribution of these granules seen in the earlier stages, especially in the ovum.
The appearance of this aggregate can be said to herald a vegetalizing influence.
As to its final fate or localization in future embryos, a remarkable series of
morphogenetic movements occur whereby these mitochondria come to lie in the
cells that will differentiate into future mesoderm and endoderm.
The mitochondrial aggregation occupies a sector of over 60°. The mitochondria are so densely packed that they form a compact mass leaving hardly any
spaces in between. Towards the AP they gradually become less and less compact
and then merge with the general cytoplasm rather abruptly. A few yolk granules
are seen to invade this zone. The vegetal parts of all four megameres (1 A-l D)
contribute towards the whole mitochondrial mass. This can be easily seen in
serial sections and in whole embryos incubated for cytochrome oxidase. These
vegetal mitochondria stain very intensely with acid fuchsin and Heidenhain's
haematoxylin and appear as intense chocolate purple masses in oxidase preparations. In sections incubated for phosphatases, they appear as colourless,
well-defined areas at the VP. The vegetal mitochondria are also appreciably
eosinophil, and phospholipid- and RNA-positive. The aggregation has been
very useful in cytochemical studies of mitochondria.
In other regions the distribution of mitochondria appears to conform to that
of the earlier stages except that the micromeres and the lateral regions of the
megameres appear denser, there being more mitochondria. The original centripetal pattern is, however, still maintained.
12-16-cell stages. The distribution is very similar to that of the 8-cell stage.
The vegetal mass is in the same position in the macromeres 2A-2D as it was
in the 8-cell stage. The uppermost quarter of micromeres (1 a-l d) appear to be
more densely-packed while the outer lateral regions of the other micromeres and
the megameres, as usual, have more mitochondria so that the general pattern is
still the same. In the 16-cell stage the micromeres (1 a-l d) have divided and the
distribution is very similar to that seen in the 12-cell stage.
Mitochondria in slug development
565
Early blastula (24-cell stage). The mitochondrial aggregation is now in the
vegetal parts of the macromeres 3A-3D. The uppermost quartet of micromeres
appears to be slightly more densely packed. There is always a tendency towards
the accumulation of more mitochondria at the AP, especially in the blastula.
This, perhaps, has a functional significance in that the contents of the cleavage
cavity are more often expelled at the AP and the cells there are pseudopodially
very active. At this stage the cleavage cavity is seen to disappear completely for
a time, bringing all the blastomeres in close contact with one another. More
mitochondria are seen in the innermost regions of all cells except blastomere
3D, along the margins of contact.
'Meso"1-blastula (32-40+ cells). This is a very remarkable stage showing the
primary mesoderm cell (4d) being formed and drawn into the cleavage cavity.
The vegetal mitochondria are now distinctly in the megameres and a good
portion of the mitochondria that were originally in 3D is cut off into 4d. This
division of 3D into 4D and 4d occurs at about the 32-cell stage and is an oblique,
vertical, unequal division whereby 4d gets about three-quarters of the cytoplasm
of 3D. As a result a massive mesodermal cell is formed and then drawn into the
cleavage cavity with the help of pseudopodial processes extended by the micromeres (Figs. 5,20). Almost the whole vegetal half of the cell 4d is densely packed
with mitochondria with the nucleus holding a prominent and significant position
in the centre separating the mitochondrial zone from the inner, more yolky
cytoplasm. The free inner boundaries of all the other cells lining the cleavage
cavity and the pseudopodial process have more mitochondria. This suggests that
mitochondria also abound in those parts of cells that are physiologically active.
The cell 4D and the other three megameres which have still not divided are all
full of the vegetal mitochondria and they will eventually form endoderm.
In a later stage (40+ cells) when 4d is lying freely in the blastocoele the
mitochondria distribute themselves more evenly in its cytoplasm and still later
when 4d divides mitochondria are equally divided among the two mesodermal
cells.
Late blastula (100-120 cells). The mitochondrial aggregation is more dispersed
among the future endodermal cells at the VP. These are the derivatives of
4A-4D and 4a-4c. These cells still give an intense reaction for cytochrome
oxidase.
Gastrula. Only the cells containing the vegetal mitochondria seem to invaginate to form endoderm (Figs. 6, 21). The invaginating flask-shaped cells have
their outer, attenuated, vegetal borders packed with mitochondria, but not so
densely as in the earlier stages. Inside the mitochondrial zone is a region
corresponding to the neck of the flask, with linear arrays of albumen vesicles
with fewer mitochondria in between them. Further inside, occupying the flask,
are the nuclei and the denser cytoplasm filled with yolk, mitochondria and other
granules. The ectodermal cells have more mitochondria and ingested albumen
filling their peripheral cytoplasm ('ectoplasm'), and yolk and mitochondria in
566
A. H. SATHANANTHAN
their inner regions ('endoplasm'), with the nuclei intervening. In general, the
outer zones, which were once denser in early embryos, appear less dense than
the inner yolky regions, owing to the albumen. Mitochondria are, however,
found everywhere around and in between yolk granules and albumen vesicles.
The most significant feature is then the vegetal aggregation in the invaginating
endodermal cells. After invagination, as the blastopore closes up, all visible
signs of this remarkable mass of mitochondria disappear. Late gastrulae
incubated for cytochrome oxidase show no intense reaction at the VP. Only a
vague rim around the closed blastopore may be seen. In sections, the vegetal
mitochondria, now in the endodermal cells lining the archenteron, are gradually
seen to disperse to other parts of the cytoplasm, as they did in the primary
mesodermal cell. Once the aggregation has played its part in the differentiation
of the germ layers, its functions seem to be over and it gradually fades away
into obscurity.
Post-gastrula. The body and mantle regions are more densely packed while
the cephalic vesicle has comparatively fewer mitochondria per unit area, owing
to the presence of albumen in both its ectoderm and endoderm (Fig. 22). The
vegetal aggregation has completely dispersed in the endoderm and is no longer
evident. Most of the endoderm now forms the inner lining of the cephalic
vesicle. The cells are massive and have developed huge albumen vacuoles, so
much so that their nuclei and cytoplasm with mitochondria have been pushed
towards the inside and occupy but a tiny portion of the cell. Mitochondria,
however, may be seen right around albumen vacuoles, especially on the outer
borders of the cells. On the radial or lateral walls they may be found in single
file or are totally absent. Some mitochondria appear to be slightly larger than
is usual. The flattened ectodermal cells of the cephalic vesicle have numerous
albumen vesicles of assorted sizes, with more mitochondria in between and
around them than in the endodermal cells.
The endodermal epithelium which is closely apposed to the invaginating shell
gland needs special mention. Cells here are small and very dense and have
slightly more mitochondria than even the overlying cells of the shell gland and
the rest of the ectoderm. Closer examination (Figs. 7, 23) shows that these
endodermal cells have little or no albumen. They have a few yolk granules and
more mitochondria, especially towards their outer borders, which are in intimate
contact with the shell-gland epithelium. The shell-gland cells have ingested
albumen in their outer regions like all other ectodermal cells of the body and
they are fairly densely packed with mitochondria. The rest of the body and
mantle ectoderm is similar to that of the shell gland in mitochondrial distribution. The mesodermal cells have no albumen but have yolk and as usual they
look denser, there being more mitochondria. The ectodermal cells lining the
oral invagination have numerous albumen vacuoles but they have also more
mitochondria than the other cells of the cephalic ectoderm.
Mitochondria in slug development
567
(b) Phase-contrast microscopy
With positive phase the mitochondria appear as rounded, homogenous,
fairly dark (brownish) objects, some showing limited Brownian movement.
They are very numerous in the vegetal regions especially of 8-cell and 16-cell
stages and vary slightly in size. Their distribution conforms to that seen by
classical methods. In centrifuged embryos they stratify mainly in the hyaloplasm
zone, being more numerous towards the equator. A few mitochondria are also
seen in the yolky zone. This has been confirmed by classical and other methods
(see centrifugation studies).
Vital staining. The mitochondria are not stainable in early embryos. Staining
for up to 2 h does not colour the granules with the dyes used. Many of the other
cellular inclusions like Golgi bodies and lysosomes take up most of the dyes
very readily. In post-gastrula stages, however, some of the mitochondria are
stained with Janus green B, while others remain colourless. This indicates that
mitochondria may change appreciably in their physiological state during certain
stages of development and also reflects a possible heterogeneity.
(c) Enzyme activity
In Arion, cytochrome oxidase and succinic dehydrogenase activities were not
found exclusively in the mitochondria. Apart from the mitochondria which
show intense activity, larger rounded and bean-shaped Golgi bodies are also as
intensely positive (Sathananthan, 1970).
Cytochrome oxidase. Mitochondria and Golgi bodies appear intense chocolate
purple while the rest of the cytoplasm is almost colourless. The mitochondria
are immobile while the Golgi bodies are active and show vigorous movement
and can be easily distinguished from the former. The mitochondria sometimes
appear as dense clouds at the VP and around nuclei. Whole embryos are
uniformly purple brown with slightly darker rings around the nuclei. The
vegetal aggregation appears as an intense chocolate brown area in the megameres at the 8-cell and later stages.
Succinic dehydrogenase. The same inclusions that show oxidase activity are
dehydrogenase-positive. But there is apparent a slight over-all decrease in
mitochondrial positivity and comparatively fewer Golgi bodies are positive.
With neotetrazolium the cytoplasm is colourless to light purple brown, while the
mitochondria and Golgi are dark crimson red. With nitro-BT these granules are
intense purple while the cytoplasm is light purple to colourless. Nitro-BT is
definitely superior as there are no diffusion artifacts and localization is excellent.
Even subtle variations in mitochondrial shape can be appreciated.
Whole embryos are uniformly reddish brown with a purple tinge. The
vegetal aggregation in the 8-cell stage is not intensely positive although, in later
embryos, the mesodermal and endodermal cells appear a shade more intense.
36
E M B 24
568
A. H. SATHANANTHAN
The regions around the blastopore and the mouth are, however, intensely
positive.
Other staining and cytochemical reactions. Mitochondria stain intensely with
Heidenhain's haematoxylin and are feebly eosinophil. They are stained fairly
intensely with Sudan black B, which can be attributed to the presence of lipids,
chiefly their membrane-bound phospholipids. The vegetal mitochondria are
Table 1. Summary of important staining and cytochemical reactions of
mitochondria
Reference
Stain or test
Heidenhain's haematoxylin
Eosin
Acid fuchsin
Sudan black B
Vital staining
Toluidine blue
Methylene blue
Neutral red
Nile blue
Janus green B
Enzyme activity
Cytochrome oxidase
Succinic dehydrogenase
Benzidine peroxidase
Alkaline phosphatase
Acid phosphatase
Other reactions
Periodic acid-Schiff (PAS)
Best's carmine
H ale's reagent
Aldehyde fuchsin/alcian blue
Pyronin Y
Ribonuclease/pyronin Y
Feulgen
Iron
Calcium
Melanin
Mitochondria*
4- + +
Metzner & Krause (1928)
McManus' method (Pearse, 1961)
Baker (1958)
+t
Burstone's method (Pearse, 1961)
Nachlas' method (Pearse, 1961)
Van Duijn's method (Pearse, 1961)
Fredricsson's method (Pearse, 1961)
Azo dye method (Pearse, 1961)
McManus' method (Pearse, 1961)
Pearse (1961)
Pearse (1961)
Spicer & Meyer (1960)
Kurnick's method (Pearse, 1961)
Pearse (1961)
Pearse (1961)
Pearl's method (Pearse, 1961)
McGee-Russel (1958)
Lillie's method (Pearse, 1961)
* + + + +, Very intense reaction; + + +, strong reaction; + + , moderate reaction;
+ , weak reaction; - , negative reaction.
f In post-gastrula only.
appreciably pyroninophil. Most of the pyroninophil reaction could be removed
by pretreating with ribonuclease. Mitochondria give negative reactions when
tested for peroxidase, phosphatases, polysaccharides, muco-substances, DNA,
calcium, iron and melanin (Table 1).
Centrifugation studies. In centrifuged ova, the mitochondria stratify chiefly
in the more centripetal hyaloplasm zone forming a very broad band (Fig. 10).
Mitochondria in slug development
569
The dividing line at the equator between the heavier yolky zone and the hyaloplasm is very sharply defined. The mitochondria are crowded more towards the
equator and decrease gradually in number as the oil cap zone is approached.
There is invariably an almost clear region apparently devoid of granules, just
below the fat cap. A few mitochondria, however, are also seen in the heavier
yolky zone, where they are found evenly distributed between the yolk granules,
or form rows or chains round the yolk granules. Thus the stratification of these
granules is incomplete.
When stratified eggs are incubated for cytochrome oxidase, the sites of enzyme
actively correspond closely to the distribution of mitochondria. The hyaline
zone gives an intensely positive reaction, the region above the equator being
most intense. On closer examination the mitochondria in the hyaloplasm zone
are the chief sites of enzyme activity. Those in the yolky region and the Golgi
bodies there are also positive. Similar, though less striking results are obtained
on incubation for succinic dehydrogenase.
(d) Ultrastructure
The mitochondria were found to show remarkable differences in fine structure
during very early development. The mitochondria in early ova (just prior to the
second maturation division) are fairly dense and their internal structure is rather
indistinct (Figs. 25, 26). Those in the astral region are particularly dense (Fig.
24), and somewhat resemble the dense 'cytoplasmic bodies' seen in mammalian
eggs (Hadek, 1965). The distribution of mitochondria in the astral zone closely
corresponds to that seen under the light microscope. Mitochondria and other
granules are generally excluded from the inner astral zone. There are, however,
a few mitochondria forming a ring round the centre of the aster, while a few
others are seen between the inner astral rays. The outer astral rays extend into
the granular cytoplasm, where they are seen to disappear between many mitochondria and yolk granules. The mitochondria associated with the aster are also
smaller (about 0-3/t) than those in other parts of the egg. In those mitochondria
which are larger and more electron-lucid a few cristae are seen projecting into
the matrix or extending right across the organelle rather haphazardly. Dense
intramitochondrial granules are rarely seen. In maturing ova the mitochondria
are mostly spherical or oval in shape, measuring about 0-5-0-6/A in maximum
diameter. Very rarely, elongate forms are seen reaching about \/i in length. The
mitochondria in mature ova have a better-defined internal structure and are
not so electron-dense (Fig. 27). The cristae are more abundant, fairly distinct
and often extend right across the granule, transveisely, diagonally or very rarely
longitudinally. Intramitochondrial granules are seen in a few cases. There is an
abundance of mitochondria in the subcortical zone confirming the lightmicroscope observations. A few are seen very close to the plasma membrane
(about 0-1-0-2/A from it). The yolk granules, Golgi bodies and lipid droplets are
seen some distance away from the surface.
36-2
570
A. H. SATHANANTHAN
27
Mitochondria in slug development
571
In the 8-cell stage (Figs. 28-31) mitochondria are highly polymorphic and
very queer in appearance. This stage, as we have already seen, is of great
morphogenetic importance in the development of An'on, when an increase in
mitochondrial number and an extraordinary segregation of mitochondria takes
place at the vegetal pole. These polymorphic mitochondria could well be those
from the vegetal aggregation. Mitochondria are extremely abundant in certain
regions and some are enormous, reaching a maximum diameter of about 1JLI.
The smaller mitochondria are roughly rounded or oval and resemble those seen
in mature ova, while the larger ones are mostly irregular in shape, there being
spherical, triangular, club-shaped, rod-like and dumb-bell shaped forms. On
the whole they are very irregular in size and shape and are comparatively larger,
almost twice or thrice as large as those seen in the ovum. The presence of
elongate, dumb-bell-shaped forms (Fig. 29) may strongly suggest that they
divide by a process akin to binary fission. Most mitochondria have an elaborate
system of well-defined cristae, often irregularly spaced within their matrices. In
others the cristae are more compacted and mote regularly arranged—transversely, diagonally or longitudinally—and stretch across the entire organelle,
leaving little space for matrix. Denser granules are found in the matrix or are
sometimes seen attached to the cristae or inner membrane.
Another salient feature is that ergastoplasmic cisternae and vesicles are often
intimately associated with mitochondria and yolk granules. Certain mitochondria (Figs. 29, 30) have vesicular elements of the endoplasmic reticulum closely
apposed to their outer membranes, sometimes enveloping the whole granule.
In others, elongate cisternae seem to extend from one granule to another. Such
an association was also seen in a few cases in ova, but was not so evident.
DISCUSSION
Morphogenetic significance. From the foregoing detailed survey of mitochondria, where over fifty embryos have been studied in serial sections by
classical methods and numerous others by vital, cytochemical and ultrastructural
Fig. 22. Sag. L.S. post-gastrula (Helly/Metzner).
Fig. 23. Shell-gland region of post-gastrula (above) at higher magnification.
FIGURES
24-31
Electron micrographs of early embryos showing fine structure of mitochondria.
Fig. 24. Small dense mitochondria from the astral region of an ovum at second
maturation, x 30000.
Figs. 25, 26. Larger less-dense mitochondria from the general cytoplasm of an ovum
at the second maturation division, x 30000.
Fig. 27. Mitochondria of a mature ovum showing better internal organization,
x 30000.
572
A. H. SATHANANTHAN
Fig. 28. Highly polymorphic mitochondria from a blastomere of an 8-cell stage,
x 9000.
Figs. 29-31. Polymorphic mitochondria (above). Note intimate association of
ergastoplasmic cisternae and vesicles with mitochondria and yolk, x 30000.
Mitochondria in slug development
573
methods one striking and illuminating fact emerges, namely that the mitochondria seem to play a very dramatic morphogenetic role in the early development of Arion.
In invertebrate development there are many instances where mitochondria
have been found to be involved with the process of early development (Raven,
1958a; Brachet, 1960; Novikoff, 1961; Gustafson, 1965). Mitochondrial
segregation, in particular, seems to be an important morphogenetic factor in
most cases.
In molluscs, segregation of mitochondria has been reported in a few cases.
Reverberi (1958) followed the distribution of mitochondria in Dentalium and
demonstrated an accumulation of these granules in the polar lobe. Polar lobes,
as we know, play an important part in morphogenesis and if removed cause
abnormalities. The polar lobe mitochondria are always retained in the D
quadrant and finally pass into the cell 4d which forms the mesoderm. These
mitochondria may be likened to the vegetal mitochondria in Arion. A distinct
segregation of mitochondria during early development was also reported in
Sphaerium (Woods, 1932) and this was later traced to cell 4d. The distribution
of mitochondria in cleavage and gastrula stages oiLimnaea (Raven, 1945, 1946)
is somewhat similar to that seen in Arion. That in the ovum, however, differs
very markedly, for the animal pole plasm was not seen in Arion. A vegetal
aggregation was also absent in Limnaea.
In the egg of the annelid Tubifex mitochondria accumulate in the polar
plasm, which was shown to have great morphogenetic significance by centrifugation experiments. These mitochondria were traced later to somatoblasts
2d and 4d (Lehmann, 1956, 1958; Lehmann & Mancuso, 1957).
Considerable work has also been done concerning mitochondria in ascidians
(Ries, 1937, 1939; Berg, 1956, 1957; Reverberi, 1956, 1957). From all their
observations it is evident that the posterior blastomeres (megameres) of 4- and
8-cell stages require the presence and activity of mitochondria. These mitochondria seem to play a very important role in differentiation—in this case the
differentiation of mesoderm and then muscle.
Gradients. Recently Gustafson (1965) has reviewed the morphogenetic
significance of mitochondrial and other gradients in sea-urchin embryos at a
biochemical level. In the light of his work and that of Horstadius (1955) on
reduction gradients, and of many others, an interesting parallel could be drawn
in the case of Arion. Although the morphogenetic pattern is different in the sea
urchin certain general comparisons could be made with regard to mitochondrial
distribution.
In the sea urchin there are evidently two gradients in mitochondrial distribution appearing at different stages of development. The first is an animal-vegetal
(basipetal) gradient which manifests itself at the mesenchyme-blastula stage and
the second is a stronger vegetal-animal (acropetal) gradient which appears in
the gastrula. It was concluded that development is controlled by the interaction
574
A. H. SATHANANTHAN
of these two oppositely directed and mutually antagonistic gradients—a concept
outlined by Runnstrom as early as 1928.
Possible mitochondria! gradient fields in Arion. In the ovum, mitochondria
are distributed along a centripetal gradient with more mitochondria in the
periphery and gradually decreasing numbers towards the centre (Fig. 32A). The
condition is much the same in the 2-cell and 4-cell stages, although one sees a
gradual increase in the number of mitochondria in the vegetal hemisphere just
below the cortical region. Then there is the dramatic segregation of mitochondria at the VP soon after the third division which profoundly alters the existing
pattern.
Fig. 32. Hypothetical gradient fields in mitochondrial distribution in early embryos.
(A) In ovum—centripetal gradient field. (B) In 8-cell stage. (C) In gastrula. (In B
and C thin-lined triangles represent animal gradients while thick-lined triangles
represent vegetal gradients in the double gradient systems.)
If an 8-cell stage is examined very closely, forgetting for a moment that the
vegetal segregation ever existed, it is seen that the original gradient pattern is
virtually unaffected. As in the earlier stages, there are more mitochondria
towards the periphery, decreasing in number as one approaches the centre of
the embryo. This is true irrespective of the presence of a cleavage cavity and
inter-blastomeric cell membranes. The appearance of the vegetal mitochondria
merely seems to emphasize the pattern already existing in that region of the
embryo. Owing to the formation of the cleavage cavity nearer the AP the
micromeres have more mitochondria as they have inherited most of the densely
packed cytoplasm that was originally in the outer upper half of the ovum. The
macromeres have inherited most of the inner core of cytoplasm with fewer
mitochondria, and the rest of the dense outer cytoplasm and of course the
vegetal aggregation. The eccentric position of the cleavage cavity, then, would
appear to suggest that there is now an animal-vegetal gradient, whereas it is in
reality a part of the original centripetal gradient field. The lateral outer parts of
the macromeres also show more mitochondria and are as densely packed as the
micromeres. With the appearance of the vegetal mass of mitochondria one may
now visualize a stronger vegetal-animal gradient opposing the original centripetal gradient field, whose centre of focus has now shifted very slightly towards
the AP and seems to be exerting its influence as if it were an animal-vegetal
Mitochondria
in slug development
575
gradient. This interpretation brings Arion more or less in line with the echinoderms.
It must, however, be reiterated that, in this context, the so-called animalvegetal gradient is really a centripetal gradient field with its centre of activity
extending throughout a curved surface, which is the subcortical zone of the
animal seven-eighths of the embryo (Fig. 32B). This indeed is the region that
will eventually differentiate for the most part into ectoderm. So regarding this
as an animal-vegetal gradient is justified. The vegetal-animal gradient, however,
is more axial in nature.
In later stages, especially in the invaginating gastrula, the two gradients can
be visualized in the more familiar manner (Fig. 32C). There are as before the
two mutually antagonistic gradients, animal and vegetal, represented as in the
case of the echinoderms.
Raven (19586) has also elucidated the influence of gradient fields in the
development of Linmaea and he concludes that the organization of the egg is
governed by the interaction of axial and cortical gradient fields.
Whatever the interpretation may be, the mitochondrial aggregation which
manifested itself after the third cleavage has obviously a vegetalizing influence
and undoubtedly has a precise morphogenetic function in Arion. It is very likely
that animalizing and vegetalizing influences interact with one another to bring
about differentiation of the germ layers, as in Limnaea and in the echinoderms.
Mitochondrial populations. The number of mitochondria seems to be more or
less constant during very early cleavage, although a slight increase may be noted
prior to the third division. At the 8-cell stage there is a dramatic rise in the
population due mainly to the appearance of the vegetal mass. An increase in
mitochondrial number during early cleavage has also been reported in Limnaea
(Raven, 1958a). As to the origin of the vegetal mitochondria, many of them
seem to have originated at the VP and appear to radiate upwards, towards the
AP. It is also likely that at least some of them arose in the subcortical zone of the
vegetal hemisphere in the earlier stages and later migrated towards the VP. As
elongate dumb-bell-shaped forms were seen in electron micrographs of mitochondria, it is probable that they arose from pre-existing mitochondria by
growth and fission. The recent demonstration of DNA in mitochondria in a
variety of cells, both adult and embryonic (Nass, Nass & Afzelius, 1965), lends
support to this view. However, the possibility that at least some mitochondria
could have originated from the cell membrane (Robertson, 1964) needs to be
carefully examined.
Between the 8-cell and the blastula stage there seems to be hardly any rise in
the mitochondrial population. The general crowding beneath the cortical zone,
seen in the earlier stages, gradually disappears in blastulae and gastrulae. The
vegetal mitochondrial population does not seem to change substantially and
these mitochondria are distributed among the future mesoderm and endoderm
cells. At gastrulation there is considerable growth, and tests for RNA suggest
576
A. H. SATHANANTHAN
that there is an active period of protein synthesis during imagination. Although
the embryo has taken in appreciable amounts of albumen the cells are still
fairly densely packed. So it is possible that there is a slight over-all increase in
mitochondrial number at this stage. A rise in the mitochondrial population is
also seen in the sea urchin at the onset of gastrulation (Gustafson, 1965). After
gastrulation copious amounts of albumen are taken in, and although the embryo
grows in size the mitochondrial population seems to be much the same as in the
gastrula stage. The mitochondria are more densely distributed only in the
visceral and body regions of the post-gastrula.
Respiratory enzymes. Recent work indicates that cytochrome oxidase and
succinic dehydrogenase are almost exclusively intramitochondrial and closely
bound to the structure of mitochondria (Lehninger, 1964). In Arion, however,
Golgi bodies also show intense activity of these enzymes (Sathananthan, 1970).
Almost all the mitochondria in Arion show cytochrome oxidase activity. This
is understandable as this enzyme is the final common pathway of most oxidative
processes in the cell. There was an increase in cytochrome oxidase activity at the
VP, coinciding with an increase in mitochondrial number in that area. Whether
or not this reflects a higher rate of respiration should be determined quantitatively. In sea urchins, respiration increases during cleavage and it rises appreciably at the onset of gastrulation when an increase in mitochondrial number is
observed at the VP. Berg & Long (1964) have shown that the vegetal mitochondria are larger, have more cytochrome oxidase activity and therefore have
a more intense energy metabolism. The condition in Arion could well be similar
to that seen in echinoderms. Some of the mitochondria in the 8-cell stage are
enormous and have a complicated internal structure. This may also account for
the increase in cytochrome oxidase activity at the VP.
When 8-cell stages are incubated for succinic dehydrogenase the vegetal
aggregation of mitochondria does not show a very intense differential staining
reaction as in the case of cytochrome oxidase. There is, then, a possible heterogeneity, as was indicated by Novikoff (1961) in liver cells. Nachlas, Walker &
Seligman (1958) have found differences in mitochondrial stainability in kidney
cells using nitro-BT. Colourless mitochondria-like granules have been seen,
especially in later embryos of Arion. These observations suggest that mitochondria may not be all alike biochemically, and may show differences in
staining reactions at different times. This seems to be more likely in the case of
developing embryos where changes in number, form and fine structure have
been noted.
There seems to be some correlation between dehydrogenase activity and
Janus green stainability. This stain gives poor results with sea-urchin embryos.
The mitochondria seem to be virtually unstainable in the early embryos of
Arion. In later stages, however, some mitochondria are stainable. This again
reflects a possible heterogeneity. Gustafson (1965) has clearly shown that
mitochondrial stainability is related to their physiological state.
Mitochondria in slug development
577
Energy-linked functions. In Arion, mitochondria are frequently located near a
supply substrate such as lipid droplets or glycogen granules or around albumen
vesicles and yolk granules. This is undoubtedly associated with their metabolic
functions. Lipid droplets have been shown to be closely associated with mitochondria by cytochemical methods (Sathananthan, 1966). This has been partly
confirmed by electron microscopy (Fig. 28). More intimate associations between
mitochondria and lipid droplets have been reported in the sea urchin (Brachet^
1960, fig. 65). It is now known that mitochondria are capable of synthesizing
and oxidizing fatty acids to completion (Lehninger, 1964). Free glycogen is
found evenly distributed in the cytoplasm but accumulations are seen around
nuclei and asters and in other parts where mitochondria are abundant. Mitochondria are also known to be involved in the metabolism of other carbohydrates. This may explain why they are seen around albumen vesicles and yolk
spherules. Albumen vesicles contain lipid, galactogen and various mucopolysaccharides (Sathananthan, 1968), while the yolk granules are also chemically
complex, being glyco-lipo-protein in natuie (Sathananthan, 1970). The mitochondria do not seem to play a direct role in yolk formation in Arion, but
together with the ergastoplasm they may be involved in its breakdown and
absorption.
Mitochondria are also known to be abundant in cells or parts of cells, where
activity is intense. They were found to be more numerous in the innermost parts
of the blastomeres lining the cleavage cavity in the blastulae of Arion. An
interesting parallel is seen in the contractile vacuoles of Protozoa, which are
surrounded by a cluster of mitochondria (Mercer, 1965). The subcortical
aggregation of mitochondria in early stages is also significant where the activities
of the cell membrane are concerned. This membrane is physiologically very
active in that molecules are constantly passing in and out through it. It is also
involved in a process akin to pinocytosis (Sathananthan, 1968). This partly
explains why mitochondria are found crowded below the cortex. It is also
known that in muscle, mitochondria form tightly packed columns between the
muscle fibrils and provide energy for muscle contraction and movement. In
Arion there is considerable pulsatory and pseudopodial activity in the micromeres due partly to the active rhythmic expulsion of the contents of the cleavage
cavity at the AP. There are also precise morphogenetic movements during
gastrulation, where micromeres were found to extend pseudopodia to the
macromeres and some of these pseudopodia are involved in the actual withdrawal of the primary mesodermal cell into the cleavage cavity. Mitochondria
are found to be abundant in all parts of the micromeres which show pseudopodial activity. There seems little doubt that the activities of the cleavage cavity,
plasma membrane and the pseudopodia all require energy and it is obvious that
the ATP produced by the mitochondria provides this energy.
Infrastructure. Electron-microscope studies of embryos of Arion seem to
indicate that mitochondria do undergo changes in form and fine structure
578
A. H. SATHANANTHAN
during early development. The mitochondria in the early embryos show a
peculiar fine structure differing markedly from those of many other cell types.
They also seem to have undergone progressive changes during very early cleavage and are the only organelles that have profoundly altered during early
differentiation. An over-all increase in mitochondrial number and size and an
increase in the number of cristae have taken place by the time the 8-cell stage is
reached. Regional differences in size and fine structure of mitochondria seem to
exist in ova. Those in the central astral zone are smaller and rather dense and
have a poor internal structure, while those in other regions, including the
cortical zone, have better internal organization. So it seems likely that there are
also structural and size differences along a centripetal gradient in Arion.
Changes and regional differences in the ultrastructure of mitochondria have
been reported in certain animals. In Tubifex, Weber (1962) found an abundance
of mitochondria in the polar plasm and cortical regions. These results are
similar to those obtained in Arion. The polar plasm is of great morphogenetic
significance and could be likened to the vegetal aggregation of mitochondria in
Arion. In the sea urchin Berg & Long (1964) showed that larger mitochondria
with more cristae were found in the invaginating vegetal cells of the early
gastrula. The numerous large, polymorphic mitochondria seen in some micrographs of the 8-cell stage could well be from the vegetal region. Yamamoto
(1964) has also found marked alterations in the structure of mitochondria in
fish embryos.
The enlargement of mitochondria, their increase in numbers, and the increase
in complexity of their internal organization, no doubt reflect higher levels of
mitochondrial activity. These events seem to coincide with their morphogenetic
segregation in Arion. The increase in cytochrome oxidase activity at the 8-cell
stage is also perhaps due to a higher level of energy metabolism. The intimate
association of mitochondria with vesicles and cisternae of the ergastoplasm is
further evidence of their increased activity.
Mitochondrial RNA and DNA. Recent studies have shown that mitochondria
contain appreciable amounts of intrinsic RNA (De Robertis, Nowinski & Saez,
1965). This has been observed in Arion also, especially in the vegetal aggregation
of mitochondria. Electron micrographs show well-developed cisternae and
vesicular elements of the ergastoplasm associated with mitochondria, which are,
no doubt, partly responsible for this RNA-positivity. The presence of RNA in
mitochondria may be very significant in the context of protein synthesis. The
generally accepted view is that mitochondrial enzyme proteins are synthesized
by the ribosomes attached to the endoplasmic reticulum and later incorporated
in mitochondria. Recent biochemical work, however, suggests that mitochondria may be able to synthesize some of their own membrane proteins (Lehninger,
1964). The discovery of intramitochondrial DNA in many types of cells with
the electron microscope (Nass et al. 1965) makes it seem more likely now that
mitochondria may be in fact sites of protein synthesis. Studies on intramito-
Mitochondria in slug development
579
chondrial DNA were not made in Arion. However, if mitochondria prove to be
universally DNA-positive they could be self-reproducing organelles and they
could also play a part in the hereditary process. It is then conceivable that they
are even better equipped to perform a morphogenetic function in developing
embryos.
Possible inductive phenomena. On account of their probable morphogenetic
significance there is reason to suppose that mitochondria, together with nucleic
acids, may play a part in induction. In gastropods, the formation of the shell
gland is thought to be due to the inductive action exerted by the tip of the archenteron on the ectoderm, with which it makes intimate contact (Raven, 1964).
In Arion, the small-celled endoderm at the tip of the archenteron has comparatively more mitochondria than the invaginating shell-gland epithelium.
Mitochondria were found to be slightly more numerous on either side of the
margins of contact of the two epithelia especially in the endoderm cells. Although
little is definitely known about the mechanism of induction it is thought that
certain chemically active substances (inducing agents) diffuse from the endoderm
cells into the ectoderm cells. The mitochondria may perhaps contribute some of
these substances in the form of enzymes or they could produce the energy for
the inductive process in the form of ATP and play a part in active transport of
these substances from cell to cell.
In this context it is also worth mentioning certain observations on the early
blastula of Arion. The 24-cell stage is unique in that the cleavage cavity completely disappears for a time, bringing all the cells together in the centre of the
embryo. There is intimate contact between the micromeres and macromeres and
this happens just before gastrulation. Curiously enough, mitochondria are
found to be more numerous in the innermost regions of all the blastomeres
(except 3D), where there is mutual contact. In addition there is an accumulation
of free RNA in the zones of mutual contact (Sathananthan, 1966). It appears
that all the blastomeres, so to speak, meet in the centre as if to decide the fate
of one of their fellows, which presumably is cell 3D, which soon buds off the
primary mesoderm cell. It is plausible that all this points towards an additional
chemical inductive process or interaction between cells in the central zone whereby the fate of 3D and perhaps that of the other macromeres are decided. Of
course, the main morphogenetic influence is exerted by the vegetal aggregation
of mitochondria, which interacts with other animal influences in the gradient
system. In fact, when the cells come together the animal and vegetal influences
can interact with each other more effectively than when there is a fluid-filled
cleavage cavity between them. Thus both intracellular and intercellular interactions could well be finally responsible for the differentiation of mesoderm and
endoderm.
In conclusion, from all the above considerations it is evident that in Arion
there seems to be a straightforward case for the morphogenetic significance of
mitochondria. In addition to their energy-linked functions they seem to play
580
A. H. SATHANANTHAN
specific directive roles in development. Segregation of mitochondria in particular
seems to be an important factor in morphogenesis. Even if differential distributions of mitochondria are normally attributed to levels of general metabolic
activity, it has been shown beyond reasonable doubt that the vegetal aggregation
of mitochondria plays a precise directive role in Arion. Many avenues available
to a better understanding of their morphogenetic significance have been
explored. But a quantitative biochemical approach and more experimental
studies are necessary before the exact significance of mitochondria in morphogenesis is fully appreciated.
RESUME
Etude des mitochondries au cours des phases precoces du
developpement chez la limace Arion ater rufus L.
La structure, la distribution, la nature cytochimique et la signification physiologique des
mitochondries ont ete etudiees au cours de l'embryogenese de la limace Arion depuis Poeuf
jusqu'au stade postgastrulaire.
Les mitochondries semblent subir des changements progressifs mais profonds dans leur
forme et leur structure fine au cours des premieres phases du developpement et leur distribution s'etablit suivant un gradient qui ressemble plus ou moins a celui de l'Oursin.
L'evenement le plus significatif de la cyto-differenciation est l'apparition d'une extraordinaire aggregation de mitochondries au stade des 8 blastomeres et suggerent une influence
vegetalisante temoignant sans nul doute d'une fonction morphogenetique tres precise. Cette
aggregation vegetative est entrainee dans une remarquable serie de mouvements morphogenetiques et ses mitochondries sont ulterieurement incorporees dans les cellules qui se
differencient en mesoderme et endoderme. Done, a cote de leur role energetique, les mitochondries semblent jouer un role directeur, de facon dramatique, dans le developpement et,
en particulier, la segregation mitochondriale semble etre un facteur important dans la
differentiation.
Certains aspects de leur distribution et de leur composition enzymatique sont discutes en
relation avec leurs fonctions metaboliques normales.
La signification du RNA et du DNA mitochondrial pour la synthese des proteines et la
duplication des mitochondries, ainsi que les roles possibles des mitochondries dans les
phenomenes d'induction sont egalement discutes a la lumiere des travaux recents.
I am deeply indebted to Professor Alastair Graham, D.Sc, and Dr Vera Fretter, D.Sc.,
for providing the facilities for my work at the University of Reading, England, and for their
invaluable guidance, constructive criticism and encouragement.
I am also grateful to Dr John R. Baker, F.R.S., for helping me a great deal with electron
microscopy at the University of Oxford; and to Professor T. Gustafson of the University of
Stockholm, Sweden, for his valuable advice.
My thanks are also due to Mr J. M. McCrae, Cytological Laboratory, Oxford University,
for technical assistance in electron microscopy; and to the Department of Sedimentology,
University of Reading, and to Mr C. H. Chang of the Science Faculty, University of Ceylon,
Colombo, for photographic assistance.
Mitochondria in slug development
581
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(Manuscript received 1 December 1969)