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/ . Embryol. exp. Morplu, Vol. 14, Part 1, pp. 25-35, August 1965
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The development of the spinal cord examined
by autoradiography
by ALEXANDER MARTIN1 and JAN LANGMAN2
From the Department of Anatomy, McGill University, Montreal
WITH 3 PLATES
T H E wall of a recently closed neural tube is generally considered to consist of
three concentric layers. Surrounding the lumen is found the ependymal zone
characterized by large pale cells known as the germinal cells of His (1889).
These cells are believed to give rise to the neuroblasts and some of the spongioblasts. Soon after formation the neuroblasts move away from the lumen to
enter a densely packed nucleated zone referred to as the mantle layer. This
layer consisting of spongioblasts and neuroblasts ultimately gives rise to the
gray matter of the spinal cord. The outermost layer, known as the marginal
layer, contains the fibres of the developing neuroblasts and later forms the
white matter.
In 1897 Schaper suggested that the primitive neural wall should not be divided
into different layers. In his opinion the dividing cells at the lumen and the nondividing ones in the nucleated area (mantle layer) belonged to the same epithelial
cell type. A similar theory was proposed by Sauer (1935, 1936, 1937), who
noticed that the cells of the so-called 'mantle layer' migrated toward the lumen
to undergo mitosis and then returned to the deeper nucleated zone. He suggested
that the dividing cells at the lumen and those located in the nucleated zone
belong to the same cell type. Furthermore, as he noticed that all the cells in the
wall of a recently closed neural tube are attached to each other at the lumen by
terminal bars, he suggested that they form a pseudo-stratified epithelium. This
interpretation was accepted by Kershman (1938), Smart (1961) and a few other
investigators, but has not been referred to in most of the presently used embryology and neuroanatomy textbooks.
Recent experiments with autoradiographic techniques have provided Sauer's
work with considerable support (Sauer & Walker, 1959; Sidman, Miale &
Feder, 1959; Kallen & Valmin, 1963; Fujita, 1963). Tritiated thymidine applied
1
Author's address: Department of Biological Structure, University of Washington Seattle,
Washington, U.S.A.
2
Author's address: Department of Anatomy, University of Virginia, Charlottesville (Va.),
U.S.A.
26
A. MARTIN and J. LANGMAN
to embryos shortly after closure of the neural tube showed DNA-synthesis to
occur exclusively in the densely nucleated zone, the so-called mantle layer.
Subsequently the labelled cells were found to migrate toward the lumen where
they divided, and then to return to the deeper layers. Hence, the cells in the
nucleated zone, as well as those near the lumen, appear to belong to the same
cell population.
As the classical delineation of the ependymal, mantle and marginal layers
apparently is no longer valid, the present experiments were undertaken to
redefine the different layers of the neural tube and to determine the origin, fate
and characteristics of their cells by means of autoradiography.
MATERIALS AND METHODS
Twenty groups of 100 chick embryos varying in age from 1 to 9 days were
treated with tritiated thymidine (200 ^c./g. estimated embryo weight) through
an opening in the shell. After closure of the shell, the eggs were returned to the
incubator and the embryos removed from 30 min. to 3 days later. The embryos
were fixed in Bouin and embedded in paraffin. Serial sections of 3-4 ^ were
mounted and stained with hematoxylin and ethyl eosin. Autoradiographs were
prepared by the coating technique of Kopriwa & Leblond (1962), using Kodak
NTB 3 emulsion. After a 3-10 days exposure time, the sections were developed,
fixed and mounted according to regular histological techniques.
In later experiments approximately two hundred 1-2 day old embryos were
treated with a second dose of thymidine-H3 2, 3 or 4 hr. after the initial treatment. These embryos were fixed after an additional 1-hr, incubation period
and then treated as above.
It is generally assumed that thymidine, a specific precursor of DNA, is only
incorporated by the nuclei which are duplicating their DNA content in preparation for cell division (Lamerton & Fry, 1963). Though in mammals
labelled thymidine remains available for incorporation for approximately 30-60
min., in early chick embryos in which the vascular system has not yet developed,
we found it to remain available from 2-3 hr. When the vascular system is well
developed, labelled thymidine remains available for incorporation for at least
24 hr. and presumably even longer.
RESULTS
Neural groove stage
A cross section through the neural groove of a 12-somite embryo treated with
thymidine-H3 1 hr. before fixation is represented in Plate 1, Fig. A. In analogy
with the terminology used for the closed neural tube, the zone bordering the
future lumen is referred to as the inner zone and that bordering the external
limiting membrane as the outer zone. The labelled nuclei, forming 60-75 per
J. Embryo!, exp. Morph.
Vol. 14. Part I
FIGS. A-D. Autoradiographs of cross sections through the neural groove of embryos varying
in age from 10-14 somites (x 440). (A) One hour after treatment. The labelled nuclei are
in the outer zone and the unlabelled ones in the inner zone of the neural wall. (B) Four
hours after treatment. The labelled nuclei are mainly in the inner zone bordering the
lumen and the unlabelled (arrows) intermingled with some weakly labelled ones in the
outer zone bordering the external limiting membrane. (C) Eight hours after treatment.
The labelled cells are once again in the outer zone. (D) Four hours and 1 hr. after treatment.
All the nuclei in the neural wall are, with a rare exception, labelled.
A. MARTIN and J. LANGMAN
(Facing page 27)
Development of the spinal cord
27
cent, of the total number of nuclei in the neural wall, are found primarily in the
outer zone, with an occasional one closer to the lumen. With the latter exception,
no label is found in the nuclei of the inner zone. Since only nuclei in the DNAsynthesis stage are able to incorporate tritiated thymidine, it may be concluded
that the outer zone contains DNA-synthesizing nuclei. Accordingly the inner
zone must contain nuclei which are in the post-duplication stage, in mitosis, or
have just completed division. The latter nuclei are either in the pre-duplication
stage leading to the next mitosis or have lost their ability to divide entirely.
In a similar embryo examined 4 hr. after treatment all the nuclei in the inner
zone, including the mitotic figures, are labelled. The unlabelled nuclei, forming
10-15 per cent, of the total number of nuclei in the neural wall, are found in the
outer zone (Plate 1, Fig. B). They must be either in the pre-duplication stage
(and therefore unlabelled) or in the DNA duplication stage but unable to
incorporate tritiated thymidine, since it is no longer available. A third possibility
is that the unlabelled nuclei have lost their ability to divide entirely. These
results suggest that the nuclei, which have incorporated thymidine-H3 in the
outer zone a few hours previously, have migrated in the meantime towards the
inner zone where they undergo mitosis (at this stage of development mitotic
figures are found exclusively along the inner surface). Simultaneously, the
unlabelled nuclei previously found in the inner zone have migrated toward the
outer zone, where they intermingle with some labelled nuclei which have not as
yet migrated inwards.
In a 14-somite embryo examined 8-10 hr. after treatment with thymidine-H3,
all the nuclei in the outer zone are labelled, while a few unlabelled ones
(approximately 10 per cent, of the total number of nuclei) intermingled with
some weakly labelled nuclei are found in the inner zone (Plate 1, Fig. C).
These results suggest that following mitosis the labelled nuclei return to the
outer zone and are replaced by some unlabelled ones.
Our experiments thus show that the nuclei in the neural wall synthesize DNA
in the outer zone (Plate 1, Fig. A), then migrate toward the inner zone to
undergo division (Plate 1, Fig. B) and subsequently return to the outer zone
(Plate 1, Fig. C).
To determine whether the unlabelled nuclei seen in Plate 1, Fig. B are in
either the pre-duplication or the duplication stage, or have lost the capacity to
synthesize DNA entirely embryos were treated with two doses of thymidine-H3,
given 4 hr. and 1 hr. before fixation. A cross section through the neural groove
of such an embryo is shown in Plate 1, Fig. D. All the nuclei in the inner and
outer zone (with the exception of 1 or 2 per cent.) are labelled, indicating that at
this stage of development all the cells of the neural wall are capable of synthesizing DNA. Furthermore, it may be concluded that the unlabelled nuclei in
Plate 1, Fig. B had failed to incorporate thymidine-H3, since the tritiated
thymidine apparently was not available. As in the neural groove stage all the
cells of the neural wall seem to behave similarly, they are thought to belong to
28
A. MARTIN and J. LANGMAN
the same cell type which will be referred to as germinal cells. The layer composed of these cells is therefore referred to as the germinal layer. It is evident
that during invagination of the neural groove, cell proliferation leads to an
increase in the number of germinal cells resulting in thickening and lengthening
of the germinal layer, but fails to produce any other cell types.
Neural tube stage
When a 22-somite embryo with a closed neural tube is examined 1 hr. after
treatment with thymidine-H3, the labelled nuclei are seen in the outer half of
the wall (Plate 2, Fig. E). Contrary to the neural groove stage in which the
labelled nuclei border the external limiting membrane (Plate 1, Fig. A), they are
now separated from it by a row of unlabelled cells. These cells are characterized
by a large round nucleus with pale nucleoplasm and a dark staining nucleolus.
Based on their histological appearance, as well as on their inability to synthesize
DNA, they are thought to belong to a different cell population, that is, the
population of neuroblasts. They arise by division of the germinal cells and form
the first representatives of the mantle layer.
To examine whether all the cells in the germinal layer still have the capacity
to synthesize DNA, 20-somite embryos were treated with thymidine-H3 4 hr.
and 1 hr. before fixation. Figures F and G (Plate 2) show that all the nuclei
in the germinal layer are labelled, while on the contrary the neuroblasts are
unlabelled. It is thus evident that at this stage of development cell proliferation
in the germinal layer gives rise to germinal cells, as well as to neuroblasts. As
the newly formed neuroblasts must migrate from the lumen to the mantle layer,
it is obvious that the cells in the germinal layer no longer form a homogeneous
population.
When a 3-day embryo (33-somites) is treated with thymidine-H3 1 hr. before
fixation, labelled nuclei are found exclusively in the outer zone of the germinal
layer, indicating that cell proliferation is still restricted to the germinal zone
(Plate 2, Fig. H). None of the cells in the mantle layer has incorporated tritiated
thymidine. Outside the mantle layer and particularly in the ventral portion of
the tube an acellular fibrous zone is now visible. This is the first appearance of
the marginal layer. Four and five-day-old embryos examined 1 hr. after
treatment show a picture similar to that of Plate 2, Fig. H: a germinal layer
with many labelled cells, an unlabelled mantle layer, and an acellular marginal
layer.
When 5-day-old embryos are examined 1, 4, 8, 12 and 24 hr. after thymidine-H3 is applied, the labelled nuclei are at first found in the outer zone of the
germinal layer, at 4 hr. close to the lumen and at 8 hr. again in the outer zone.
At 12 hr., however, one group of labelled cells is located in the germinal layer
close to the lumen while another much smaller group is migrating among the
unlabelled neuroblasts of the mantle layer. This is particularly evident in
J. Embryol. c.xp. Morph.
Vol. 14, Part I
PLATE 2)
FIG. E. Autoradiograph of a segment from the neural tube of a 22-somite embryo treated
with thymidine-H3 1 hr. before fixation (x 850). Note the unlabelled nuclei bordering the
external limiting membrane (arrows). The labelled nuclei are in the outer zone of the
germinal layer; the nuclei in the inner zone, including the mitotic figures, are unlabelled.
FIG. F. Autoradiograph of a segment from the neural tube of a 20-somite embryo treated
with thymidine-H3 1 and 4 hrs. before fixation. Note the unlabelled neuroblast nuclei
all the nuclei in the germinal layer are labelled.
FIG. G. Autoradiograph of the neural tube of the embryo represented in Fig. F (x440).
Note that the unlabelled neuroblasts form a row of cells between the external limiting
membrane and the germinal layer.
FIG. H. Autoradiograph of the neural tube of a 33-somite embryo 1 \ hr. after treatment
(x 300). The unlabelled neuroblasts form a distinct mantle layer, particularly evident in
the ventral aspect of the tube. Note that the labelled nuclei are located in the outer zone
of the germinal layer. A thin fibrous acellular zone, the marginal layer, is visible between
the mantle layer and the surrounding mesenchyme.
A. MARTIN and J. LANGMAN
{Facing page 28)
Vol. 14, Part 1
J. Embryol. exp. Morph.
PLATE 3
FJG. I. Autoradiograph of the anterior horn of a 6-day embryo, 24 hr. after treatment
( x 850). An occasional labelled neuroblast is seen deep in the mantle layer. The majority
of the germinal cells is labelled. The thick marginal zone is still acellular.
FIG. J. The anterior horn of a 6|-day embryo, 6 hr. after treatment with thymidine-H3
(x 850). Note the row of labelled cells extending from the periphery of the tube into the
mantle layer. The neuroblasts in the anterior horn are not labelled.
FIG. K. The anterior horn of an 8-day embryo, 2 hr. after treatment (x 850). Many small
labelled nuclei are seen in the marginal zone, an occasional one among the neuroblasts
of the mantle layer, and a few in the germinal zone.
FIG. L. The anterior horn of a 9-day embryo, 2 hr. after treatment (x 850). Heavily labelled
nuclei are seen in the marginal and mantle zone. None of the neuroblasts appears to be
labelled.
A. MARTIN and J. LANGMAN
(Facing page 29)
Development of the spinal cord
29
embryos examined 24 hr. after treatment (Plate 3, Fig. I). Some of the labelled
cells have now penetrated deep into the mantle layer, where they differentiate
into neuroblasts. The marginal layer is still acellular.
A 6|-day-old embryo examined 6 hr. after treatment reveals that the majority
of the labelled cells are in the germinal layer, but a few are visible in the mantle
and marginal layer (Plate 3, Fig. J). As a labelled germinal cell requires at least
8 hr. to migrate out of the germinal layer, the labelled cells in the mantle and
marginal layer must be either synthesizing DNA in situ or have migrated into
the neural tube from outside. The last possibility seems the most likely one, as
rows of labelled cells, cytologically different from those in the mantle layer,
are frequently seen to extend from the periphery of the tube into the marginal
and mantle layer (Plate 3, Fig. J). It is believed that these rows of cells represent
ingrowing vascular buds. In addition to these rows, isolated labelled cells are
seen in the marginal layer close to the surrounding mesenchyme. They seem to
have no relation to the vascular buds.
From day 7 on, the influx of labelled cells from the outside of the tube
becomes more marked (Plate 3, Figs. K and L). A large number of small dark
staining nuclei is visible in the marginal layer, while in the mantle layer groups
or isolated large densely labelled cells are present. Though some of the cells
of the latter group are associated with ingrowing blood vessels, many with
different morphological characteristics have no spatial relationship to the
vessels.
By the 9th day of development the mantle layer consists of neuroblasts
originating in the germinal layer and a number of differently shaped cells. Some
of the latter undoubtedly come from outside the tube, while others probably
come from other sources. Since during the 8th and 9th day of development the
lumen of the tube decreases considerably in size, and consequently many cells
of the germinal layer lose their attachment to the lumen, it is thought that many
of these germinal cells migrate into the mantle layer to become supporting cells.
DISCUSSION
When a recently closed neural tube is examined under the microscope, the
wall appears to consist of three different morphological zones: an inner zone
along the lumen with a few large mitotic figures; a middle zone with densely
packed oval shaped nuclei; and an outer anuclear zone. According to the
classical theory demonstrated in the drawing of Hardesty (1904), these three
zones are thought to represent the ependymal, mantle and marginal layers,
respectively. In our opinion, however, the wall of the recently closed neural
tube consists of only one layer which is formed by one cell type—the germinal
cells.
During DNA-synthesis the germinal cells are thought to extend over the full
width of the wall with the nucleus in the densely nucleated middle zone, a long
slender cytoplasmic extension toward the lumen and the bulk of the cytoplasm
30
A. MARTIN and J. LANGMAN
in the outer anuclear zone. Our observations, as well as those of Sauer & Walker
(1959); Sidman, Miale & Feder (1959); Fujita (1963); and Kallen & Valmin
(1963) indicate that DNA-synthesis occurs only in the densely nucleated middle
zone (frequently referred to as the mantle layer).
Evidence for the suggestion that the outer anuclear zone consists of cytoplasm
was found in an additional series of experiments in which chick embryos were
treated with colchicine (Martin & Langman, 1965). In areas with high mitotic
activity all the cells were arrested in the metaphase and the acellular zone was
absent. In areas with low mitotic activity the acellular outer zone was present
as in normal specimens.
Electron microscope studies have provided ample evidence that the cells
forming the neural wall have a slender cytoplasmic foot on the lumen, where they
are attached to each other by rigid terminal bars (Duncan 1957; Brightman &
Palay, 1963; Fujita & Fujita, 1963; Tennyson and Pappas, 1962). During
mitosis the germinal cells round up, and since they are fixed to adjacent cells by
terminal bars at the lumen, the nucleus and cytoplasm of the cell will be drawn
toward the lumen where division takes place. Not a single mitotic figure was
ever observed other than on the lumen.
From our double labelling experiments it is apparent that during the neural
gioove stage all the cells in the neural wall are capable of synthesizing DNA
and that no differentiation into neuroblasts occurs. Hence, all the cells composing the neural wall belong to the same population, that is, the population of
neural epithelial cells, or as we would prefer to call them, germinal cells. The
layer so composed is therefore called the germinal layer. The term 'germinal
layer' seems to us more suitable than the name 'matrix layer' introduced by
Fujita, as the latter is usually used in the histological literature to indicate the
intercellular substance of a tissue. The term ependymal layer is thought to be
slightly misleading, since at the early stages of development none of the cells
has the characteristics of an ependymal cell and, furthermore, because only an
extremely small percentage of the cells ultimately will differentiate into ependymal cells. In our opinion, the germinal cells first give rise to the neuroblasts;
secondly, with the reduction in size of the lumen, to the supporting cells, and
only those remaining attached to the central canal become ependymal cells.
Therefore the term germinal cell seems to us most appropriate. It is thus evident
from our experiments that during the neural groove stage the germinal cells
synthesize DNA in the outer zone, then move toward the lumen to divide and
finally return to the outer zone to repeat the cycle almost immediately.
Shortly after closure of the neural tube, division of the germinal cells does not
exclusively yield similar daughter cells, but also gives rise to a different cell type.
The nuclei of these cells are round with a pale nucleoplasm and a darkly stained
nucleolus in contrast to the oval shaped nuclei of the germinal cells. On the
basis of these morphological characteristics and their inability to synthesize
DNA we believe that these cells are neuroblasts.
Development of the spinal cord
31
As the neuroblasts are formed by division of germinal cells, the question
arises whether any of the dividing cells show special characteristics. In this
regard it is interesting to note that Fujita (1962) reports that the mitotic figures
in the early neural tube are found exclusively along the lumen and that the
direction of the spindle fibres is always parallel to the inner surface of the tube.
In some of our unpublished experiments, however, in which the direction of the
spindle fibres of a recently closed neural tube was examined, it was found that a
small percentage (5-10 per cent.) had their spindle fibres perpendicular to the
inner surface of the tube. Since the germinal cells are attached to each other by
terminal bars at the lumen, it is thought that when a cell divides with its spindle
fibres perpendicular to the surface, it is possible that one of the daughter cells
loses its attachment to the lumen and gains the freedom to migrate into the
mantle layer, while the other daughter cell remains attached and stays in the
germinal layer. Further studies on this hypothesis are presently undertaken.
Until the 6th day of development a continuous stream of neuroblasts leaves
the germinal zone and a distinct mantle layer is formed. This layer is particularly
evident at the ventral aspect of the neural tube but dorsally it tapers off and
consists of two to three layers of neuroblasts. Despite a careful search for the
presence of supporting cells in the ventral part of the mantle layer, none was
found. It seems probable that until the 6th day of development the germinal
cells give rise to neuroblasts but not to any supporting cell types.
After the 6th day of development, many heavily labelled cells begin to
penetrate into the marginal and mantle layers from outside the tube. They are
seen either in long rows or as isolated units. Though the rows of cells are
unmistakably mesenchymal buds developing into blood vessels, the isolated
cells have nuclei with different shapes. Some have small round dark staining
nuclei, while others, forming a much smaller group, are characterized by oval
or sometimes rod-shaped nuclei. At first these cells appear in the marginal
layer close to the surrounding membranes, but gradually they penetrate through
the marginal zone into the mantle layer. The cells with the round dark staining
nucleus, however, remain predominantly in the marginal zone. Though it is
difficult to recognize a differentiating cell by the shape and size of its nucleus
only, we would like to suggest, in analogy with the morphological characteristics
of the microglia cells, that the ones with the oval to rod-shaped nuclei represent
microglia cells. The other cell type, however, could not be identified, but is
presumably one of the supporting types.
In addition to the two above-mentioned cell types which originate outside
the tube, a third type was found particularly in the posterior aspect of the tube.
These cells had large oval nuclei without distinct nucleoli and resembled the
germinal cells. In the posterior horn they were so numerous that the neuroblasts
could hardly be found. As their appearance coincided with the reduction in
size of the lumen of the tube into the small central canal, it is thought that they
represent germinal cells which have lost their attachment and then wander into
32
A. MARTIN and J. LANGMAN
the mantle zone. Some of these wandering germinal cells differentiate into
supporting cells, while others degenerate.
It is thus evident from our experiments that during the neural groove stage
cell proliferation in the germinal zone results in the production of germinal cells
only. After closure of the tube, however, some of the dividing germinal cells
give rise to neuroblasts which form the first representatives of the mantle layer.
With obliteration of the lumen most of the germinal cells, particularly in the
posterior aspect of the tube, lose their attachment to the lumen and wander
out into the mantle layer to become supporting cells. The cells remaining in
contact with the lumen of the central canal then differentiate into ependymal
cells.
SUMMARY
Chick embryos ranging in age from 24 hr. to 9 days were treated with thymidine-H3 through an opening in the shell to examine the proliferation and
migration of the cells in the neural tube. One to 24 hr. after treatment the
embryos were fixed in Bouin, sectioned at 4/x and processed for autoradiography
by the coating technique. The following observations were made:
1. One hour after treatment the nuclei in the outer half of the wall of the
neural groove are labelled, while those in the inner half fail to incorporate
tritiated thymidine (Plate 1, Fig. A). Three hours later the labelled nuclei are
found mainly in the inner half where they divide (Plate 1, Fig. B), while 10 hr.
later they are once more in the outer half (Plate 1, Fig. C). Hence, during
invagination of the neural tube the nuclei synthesize DNA in the outer zone,
then migrate toward the lumen where they divide and subsequently move again
toward the outer zone.
2. When embryos with an open neural groove are treated with two doses of
thymidine-H 3 ,4 and 1 hr. before fixation, respectively, all the nuclei in the neural
wall incorporate thymidine-H3, indicating that at this stage of development all
the cells are capable of synthesizing DNA (Plate 1, Fig. D). Since all the cells
behave in the same manner and no differentiation is apparent, they are thought
to belong to one and the same cell type, referred to as the germinal cells.
Furthermore, as the wall of the neural groove is formed by germinal cells only,
it is referred to as the germinal layer.
3. After closure of the neural tube a different cell type, with a round nucleus,
pale nucleoplasm and dark staining nucleolus appears at the periphery of the
tube directly against the basement membrane. These cells, which originate in
the germinal layer, do not incorporate thymidine-H3 at any time during further
development. They are thought to be neuroblasts and form the first representatives of the mantle layer.
4. During the 3rd to 5th day of development cell proliferation occurs exclusively in the germinal layer while the mantle layer, continuously increasing in
size, fails to show any mitotic activity. A thin fibrous zone, referred to as the
Development of the spinal cord
33
marginal layer, begins to surround the mantle layer at the 3rd day. It remains
acellular until day 6.
5. After the 6th day of development different cell types begin, to penetrate
the neural tube from the outside. Some of these appear in rows and are believed
to be the vascular buds while others are seen as isolated cells. The latter are
characterized by small round dark staining nuclei while some have an oval to
rod-shaped nucleus.
6. By the 8th day of development the lumen of the neural tube undergoes a
reduction in size and most of the germinal cells lose their attachment to the
lumen. These cells wander into the mantle layer where they seem to differentiate
into supporting cells. The germinal cells remaining in contact with the lumen
of the central canal differentiate into ependymal cells.
RESUME
Le developpement de la moelle epiniere examine par autoradiographie
Des embryons de poulet ages de 24 hr. a 9 jours ont ete traites a la thymidine3H a travers un orifice de la coquille pour examiner la proliferation et la
migration des cellules dans le tube nerveux. Une a 24 hr. apres le traitement,
les embryons ont ete fixes au Bouin, coupes a 4{x et traites pour l'autoradiographie par emulsion liquide. On a fait les observations suivantes:
1. Une heure apres le traitement, les noyaux de la moitie externe de la paroi
de la gouttiere neurale sont marques, alors que ceux de la moitie interne n'ont
pas incorpore de thymidine tritiee (Planche 1, Fig. A). Trois heures plus tard, les
noyaux marques se trouvent surtout dans la moitie interne, ou ils se divisent
(Planche 1, Fig. B) tandis que dix heures plus tard ils sont de nouveau dans la
moitie externe (Planche 1, Fig. C).
Done, au cours de l'invagination du tube neural, les noyaux synthetisent de
l'ADN dans la zone externe, puis emigrent vers la lumiere ou ils se divisent et
en suite emigrent de nouveau vers la zone externe.
2. Quand des embryons a gouttiere neurale ouverte sont traites par deux
doses de thymidine-3H, respectivement 4 hr. et 1 h avant la fixation, tous les
noyaux de la paroi neurale incorporent de la thymidine-3H, indiquant que, a
ce stade du developpement, toutes les cellules sont capables de synthetiser de
l'ADN (Planche 1, Fig. D). Comme toutes les cellules se comportent de la
meme maniere et qu'il n'y a pas de differentiation apparente, on pense qu'ejles
appartiennent a un seul et meme type de cellules, qu'on designe sous le nom de
'cellules germinatives'. De plus, comme la paroi de la gouttiere neurale est
formee par ces seules cellules, on la designe sous le nom de 'couche germinative'.
3. Apres la fermeture du tube nerveux, un type cellulaire different, avec un
noyau arrondi, du nucleoplasme pale et un nucleole a coloration foncee, apparait
a la peripherie du tube, directement contre la membrane basale. Ces cellules,
qui naissent dans la 'couche germinative', n'incorporent jamais de thymidine-3H
3
34
A. MARTIN and J. LANGMAN
au cours du developpement ulterieur. On pense que ce sont des neuroblastes
et qu'elles forment les premiers representants de l'assise enveloppante.
4. Du 3e au 5e jour du developpement, la proliferation des cellules a lieu
exclusivement dans la couche germinative tandis que l'assise enveloppante, dont
la taille s'accroit continuellement, ne presente aucune activite mitotique. Une
mince zone fibreuse, la 'couche marginale', commence a entourer l'assise
enveloppante le 3e jour. Elle reste acellulaire jusqu'au 6e jour.
5. Apres le 6e jour du developpement, diverses sortes de cellules commencent
a penetrer de l'exterieur dans le tube nerveux. Quelquesunes d'entre elles
apparaissent en rangs et on pense qu'il s'agit des bourgeons vasculaires, tandis
que d'autres sont isolees. Cellesci sont caracterisees par des noyaux petits,
arrondis, de coloration sombre, tandis que certaines ont un noyau ovale ou en
batonnet.
6. Le 8e jour du developpement, la lumiere du tube nerveux subit une reduction de taille et la plupart des cellules germinatives perdent leur adhesion a la
lumiere. Ces cellules emigrent dans l'assise enveloppante ou elles semblent se
differencier en cellules de soutien. Les cellules germinatives restees en contact
avec la lumiere du canal central se differencient en cellules ependymaires.
ACKNOWLEDGEMENTS
This work was supported by a Term Medical Grant of the Research Council of Canada.
The advice of Dr C. P. Leblond and the skilled assistance of Dr Beatrix Kopriwa is gratefully
acknowledged. In the later phase this work was supported by a sub-grant from a National
Institutes of Health Institutional Grant to the University of Virginia School of Medicine.
REFERENCES
M. W. & PALAY, S. L. (1963). The fine structure of ependyma in the brain of
the rat. /. Cell Biol. 19, 415-39.
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{Manuscript received 18th April 1964, revised 8th February 1965)