/. Embryol. exp. Morph., Vol. 16, 3, pp. 497-517, December 1966
With 2 plates
Printed in Great Britain
497
The development of
the chick embryo diencephalon and mesencephalon
during the initial phases of neuroblast
differentiation
By KATHERINE M. LYSER 1
Harvard Biological Laboratories, Cambridge, Massachusetts,
and Hunter College of the City University of New York
The development of the nervous system presents many interesting problems
as a developing system with numerous parameters of differentiation as well as
from the point of view of the establishment of adult structure and function. With
our growing understanding of developmental processes in general, and interactions at various stages of development in particular, it should be profitable to
study more closely events of each period in a developing system, looking for
information concerning their immediate control and their relation to events of
other periods. In the nervous system, one phase which should be investigated
much more thoroughly—especially from the point of view of the control of
cellular differentiation—is that of the initial appearance of neuroblast cells
and formation of the first nerve processes. Most studies of normal embryos
which have included the period of initial differentiation have been primarily
concerned with tracing the origins of definitive nuclei and fiber tracts, though
possible mechanisms controlling various aspects of their development have of
course been discussed.
The present study is concerned specifically with the period of initial differentiation of cells and fibers in the diencephalon and mesencephalon of the chick
embryo. This region has been chosen because it is among the early areas of
differentiation, and it contains a number of different centers, which are not
continuous with other areas of differentiation at first. This study was begun
as part of a thesis (Lyser, 1960) and reconsidered in the light of recent work in
related fields.
In the chick embryo, neuroblasts with processes appear first in the hind brain
and shortly thereafter in the diencephalon and mesencephalon, where the first
neuroblasts with processes have been reported at 17- or 18-somite stages
1
Author's address: Department of Biological Sciences, Hunter College, New York,
10021, U.S.A.
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K. M. LYSER
(Tello, 1923; Windle & Austin, 1936). The initial differentiation of neurites
thus takes place quite early in the development of the central nervous system.
In the spinal cord of the chick embryo (Hamburger, 1948), and presumably
in the brain also, initial neuroblast differentiation begins while or before proliferation has reached its peak (cf. Hamburger, 1948; Tello, 1923; Windle &
Austin, 1936) and so overlaps this phase. It is of course continuous with the
later phases of development, including histological differentiation, but it begins
well before these become apparent. In the diencephalon and mesencephalon
the mantle layer does not become distinct from the inner cell layer nor can the
longitudinal columns of cells be distinguished until about the fourth day
(Palmgren, 1921; Rendahl, 1924; Kuhlenbeck, 1937).
MATERIALS AND METHODS
Forty-five 13- through 30-somite chick embryos were studied. All the embryos were serially sectioned, either sagitally or transversely, and stained for
nerve fibers with silver. Eighteen embryos were from the collection of Professor
Leigh Hoadley. These embryos had been fixed in 95 % ethanol and stained with
pre-war German Protargol by a modified Bodian (1936) method. The other
twenty-seven embryos were prepared for this study. A number of fixatives
recommended for the embryonic nervous system and several silver stains were
tried in various combinations. Staining by a modified Holmes's (1942) method
was most satisfactory. For these young embryos, the following fixatives were
found to be useful: 95 % ethanol, Bodian's fixative no. 2 or no. 4 (Bodian, 1937),
Mahdissen's fixative as given by Gray (1954, p. 192), Lavdowsky's mixture as
given by Guyer (1953, p. 236), or Lavdowsky's mixture modified by substituting
formic acid (1-6 ml) for acetic acid (2-0 ml). Embryos remained in ethanol for
H h or in one of the other fixatives for approximately 24 h. They were stored
in 70 % or 80 % ethanol, dehydrated in ethanol, cleared in cedar-wood oil, and
embedded in 60-63 °C Tissuemat (Fisher). Serial sections were cut at 10 or 12 /*.
Graphic reconstructions of some of the younger embryos were made by
drawing neuroblast cells with processes and other segments of fibers on camera
lucida tracings of each section and then tracing these on to an outline of the
brain (Text-figs. 1-3). All cells and fibers which could be seen were recorded.
In addition, diagrams of the pattern in some of the older embryos were made by
sketching representative cells and fibers on an outline of the brain as the
sections were studied (Text-figs. 4-6). In these drawings the actual number of
cells present is not indicated; only a few are shown, illustrating the locations
and orientations of the neuroblasts and fibers observed.
Initial neuroblast differentiation
499
OBSERVATIONS
There is some variation in the development of the neuroblasts and nerve
fibers among embryos of the same stage as determined by somite count. This
appears to be due to a difference in the time at which differentiation of axons
begins in this area of the brain in relation to the development of the somites.
However, development is fairly regular in general location and arrangement of
cells and fibers and in the order of their appearance. The embryos can be
arranged in sequence by considering the numbers and distribution of cells and
fibers together with the number of somites and incubation time.
The earliest stage at which fibers were identified in the diencephalon and
mesencephalon in the group of embryos studied was the 14-somite stage. For
purposes of description, development from the earliest appearance of nerve
fibers in this region through the 30-somite stage has been divided arbitrarily
into five periods: (A) 14- to 16-somite embryos in which axon differentiation in
this region is just beginning, (B) 16-somite embryos in which differentiation is
slightly more advanced, (C) 17- to 18-somite embryos, (D) 19- to 22-somite
embryos and (E) 23- to 30-somite embryos.
At the beginning of the period of development under consideration, the wall
of the neural tube is essentially a pseudostratified columnar epithelium, the
cells of which may be referred to as neural epithelial cells. Those which are
undergoing division move toward the neurocoel; the nuclei of interphase cells
are at various levels (Sauer & Walker, 1959; Sidman, Miale & Feder, 1959;
Fujita, 1963). At the outer edge is a nucleus-free zone, consisting of the outer
ends of the epithelial cells, where the marginal layer will subsequently form.
This will be referred to here as the 'peripheral zone'; the area between the
peripheral zone and the neurocoel will be called the 'nuclear zone'. Neuroblasts
and fibers that are parallel to the surface of the neural tube and oriented in a
dorso-ventral direction or obliquely will be referred to as 'circumferential',
those which are parallel to the longitudinal axis of the neural tube as 'longitudinal \ and those which are perpendicular to the margin of the neural tube
as 'radial'.
In these preparations, neuroblasts with processes stand out because the
cytoplasm is more darkly stained than the cytoplasm of adjacent epithelial cells.
Their processes and other segments of fibers are black. It usually is not possible
to trace a fiber that extends through several sections from one section to the
next, even in the younger embryos where there are only a few fibers. As with
other methods for identifying nerve cells and fibers, the question of whether all
neuroblasts are stained can be raised. It seems likely that with this method
most neuroblasts are recognizable, but it cannot be definitely determined that
all are stained. Silver methods may demonstrate only nerve cells with neurofibrillae (Guillery, 1965; Gray & Guillery, 1966), but there is no evidence at
present that there are nerve processes in the early embryo which do not contain
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K. M. LYSER
neuro-fibrillae or neuro-tubules. In electron micrographs of motor neuroblasts
of chick embryos, for example, all the axons which could be definitely identified
contained fibrillar structures (Lyser, 1964).
Two other problems encountered in studying silver-stained sections should
be remembered in regard to the intent and the basis of interpretation of observations. As indicated above, in well-stained sections nerve fibers and the cell
bodies of neuroblasts with processes are usually distinct. Sometimes, however,
the edges of epithelial cells are dark and difficult to distinguish from axons, or
fibers do not show up well and neuroblasts from which processes arise cannot
be clearly distinguished. In descriptions of individual embryos the lower
number of fibers recorded includes only those which can be identified with
certainty; the higher number also includes those cellular structures which are
thought to be neuroblasts or fibers but which are not clearly identifiable. Both
have been included in the figures.
The neuroblasts which can be seen in any one embryo represent less than
the total number present, since the plane of section must be nearly parallel to
the long axis of a neuroblast in order to see the origin of the process from the
cell body. It is difficult to see cross-sections of individual fibers if they are
scattered singly, even though groups of transversely sectioned fibers show up
well. To obtain an adequate picture of the pattern of nerve cell bodies, which
are oriented in various directions, both transversely and sagittally sectioned
embryos must of course be studied. Also, deviation of the plane of section from
a true sagittal or transverse plane must be taken into account. In 16- to 18-somite
embryos in particular, the plane of section is often at an angle to transverse or
sagittal in part of the diencephalon and mesencephalon because the cranial
flexure is beginning and the head of the embryo is turning to the right at the
same time.
Individual cells and fibers of each embryo, and the numbers present in each
case, have been analysed in order to obtain as much information as possible on
the pattern of differentiation and on the way development proceeds. Specific
numbers, etc., are not intended to have any other significance per se. To repeat,
it is felt that study of a series of embryos sectioned in different planes, and
including several embryos of each stage, gives meaningful information of
this sort.
A. 14- through 16-somite stage: initial appearance of neuroblasts with processes
The first group includes the least advanced embryos in which nerve fibers
were found in the diencephalon and mesencephalon. In each of these embryos
a few neuroblasts with processes and a few additional segments of fibers could
be seen on each side in the posterior half of the diencephalon. The embryo
illustrated (Text-fig. 1), which was sectioned sagittally, has at least 2, and
possibly 4, neuroblasts with processes and 7 other nerve fibers on the right.
There are at least 2 and possibly 5 fibers on the left, 3 of which seem to come
Initial neuroblast differentiation
501
from cells in the same sections. In other embryos studied a few more fibers are
visible; 1-4 neuroblasts and 4-20 other fibers were found on each side.
These neuroblasts and fibers are located within an area including the lower
part of the dorsal half and the upper part of the ventral half of the lateral wall,
and extending from the junction of the diencephalon and mesencephalon to
M
Text-fig. 1. Group A: 16-somite embryo, sagittal sections. Graphic reconstruction
of neuroblasts with processes (a-d, f-h) and segments of fibers (e, others unlabelled)
in the diencephalon and mesencephalon of one of the least-advanced embryos.
O, Location of optic stalk. Arrows indicate boundaries of telencephalon (7),
diencephalon (D) and mesencephalon (M). A, Right side; B, left side, x 110.
about the middle of the diencephalon. The area covered is not quite as large in
the least-advanced embryos (Text-fig. 1) as in those with slightly more neuroblasts and fibers. The individual neuroblasts and fibers are scattered singly
among the neural epithelial cells. There is no indication of a specific cell by cell
pattern of distribution within this area. The more ventrally placed neuroblasts
in this group appear comparable to those identified by other authors (Tello,
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K. M. LYSER
1923; Windle & Austin, 1936) as belonging to the future nucleus of the medial
longitudinal fasciculus. The more dorsal neuroblasts, certainly in slightly older
embryos if not at the earliest stages, are farther dorsal than the early cells described by these authors, and apparently correspond to cells they identify
as thalamo-tegmental or thalamo-bulbar.
The neuroblasts in these embryos are oriented circumferentially with processes extending ventrally (Text-fig. \a:c, d; \b:f,g; Plate 1, fig. A), or radially,
with their long axes parallel to adjacent epithelial cells and their processes
extending laterally (Text-fig. 1 a:b; lb:h). There are also some neuroblasts at an
angle between dorsal-ventral and medial-lateral orientations (Text-fig. I a:a).
Due to the orientation of the cell body and axon arising from it relative to the
plane of section, the radially oriented neuroblasts can be seen best in transverse
sections and the circumferential neuroblasts in sagittal sections. All of the cell
bodies of the radially oriented cells and most of those of the circumferential
cells are in the nuclear zone. A few circumferential cells are just outside the
nuclear zone.
The axons of almost all of the neuroblasts grow ventrally or slightly obliquely
in a ventral and posterior direction. Those of the radially oriented cells, which
initially grow laterally, turn ventrally within the nuclear zone, at the border
between the nuclear zone and the peripheral zone, or in the peripheral zone
(Text-fig, la: b). Fibers that extend ventrally within the nuclear zone tend to
enter the peripheral zone eventually. The fibers in the peripheral zone lie in the
inner or middle part; very few are found along the outer edge. Occasionally
branching fibers are seen (Text-fig. 1 b:e). In these embryos the fibers whose cell
bodies are not seen appear as short, straight or slightly wavy segments, radially
or circumferentially oriented. In the more advanced embryos some are slightly
longer than those of the younger embryos. Occasionally a few fibers oriented
in a longitudinal direction are seen in a ventral position at the junction of the
diencephalon and mesencephalon.
B. 15- through 16-somite stage: more advanced embryos
During this period more neuroblasts are added to the original area of differentiation and longitudinal fibers appear ventrally. In the more advanced embryos
of this group (Text-fig. 2), cells with processes and fibers can be seen farther
dorsally, anteriorly and ventrally in the diencephalon than previously and also
in the anterior mesencephalon, especially in the ventral part. The distribution
of neuroblasts and fibers is more dense than before, particularly at the center of
the area, where the first neuroblasts were located.
The neuroblasts in the lateral diencephalon are oriented in a radial or circumferential direction as the first ones were. The cell bodies are usually located
in the nuclear zone, and most of the processes extend laterally, ventrally or
obliquely in a posterior and ventral direction (Text-fig. 2; Plate, 1 fig. B). Some
of these processes turn. An axon arising from the posterior-lateral side of the
Initial neuroblast differentiation
503
cell body may turn ventrally, immediately (Text-fig. 2: a) or at the outer edge
of the nuclear zone (Text-fig. 2:b, c). A ventral process may turn posteriorly
(Text-fig. 2:d) or laterally (Text-fig. 2:e). A few axons can be seen to branch
(Text-fig. 2:b,f, g). For example, one of these (Text-fig. 2:/) ends at the edge
of the nuclear zone in a triangular enlargement with what seem to be very fine
branches curving out dorsally and ventrally. Another (Text-fig. 2:g) divides
just before reaching the outer edge of the nuclear zone; the two branches curve
around the opposite sides of another cell. One cell (Text-fig. 2:b) extends
M
Text-fig. 2. Group B: 18-somite embryos, sagittal sections. Graphic reconstruction
of neuroblasts with processes (a-g, i-j) and segments of fibers (h, others unlabelled)
in the diencephalon and mesencephalon, right side. O, Location of optic stalk.
Arrows indicate boundaries of telencephalon (T), diencephalon (D) and mesencephalon (M). x 150.
laterally to the outer edge of the nuclear zone, where it runs ventrally a short
distance, then turns in a lateral direction and ends in a Y-shaped branch. Most
of the segments of fibers which can be seen in this area (Text-fig. 2) are oriented
in a dorsal-ventral direction or obliquely from anterior and dorsal to posterior
and ventral.
At the ventral edge of the original area in the diencephalon and in the
anterior mesencephalon, longitudinal fibers, as well as a few which are radially
or circumferentially oriented, are seen. Some of the longitudinal fibers seem
to be processes of neuroblasts located in the lateral part of the diencephalon.
This is suggested by the fact that a few of the most ventral circumferential
504
K. M. LYSER
fibers turn posteriorly among the longitudinal fibers (Text-fig. 2: h) or occasionally anteriorly. Other longitudinal fibers can be seen to arise from neuroblasts
located at this level, often in the nuclear zone. The processes extend posteriorly,
or posteriorly and laterally, from the cell bodies and pass into the peripheral
zone, where they continue in a posterior direction (Text-fig. 2:i). A few of the
neuroblasts in the mesencephalon have processes which extend in a ventral
direction (Text-fig. 2:j). As in other areas, a few of the fibers branch.
M
Text-fig. 3. Group C: 18-somite embryo, sagittal sections. Graphic reconstruction
of neuroblasts with processes and segments of fibers in the diencephalon and mesencephalon, left side. O, Location of optic stalk. Arrows indicate boundaries of
telencephalon (T), diencephalon (D) and mesencephalon (M). x 150.
C. 17- through 18-somite stage
Between the 16- and 18-somite stages, there is further development of the
first area of axon differentiation, and neuroblasts in a second area, the dorsal
mesencephalon, begin to send out processes. The latter are tectal cells.
The number of neuroblasts in the lateral diencephalon continues to increase
and new cells with processes appear anterior and posterior to those seen previously. By the 18-somite stage, nerve fibers and neuroblasts are found from
the level of the optic stalk to the anterior edge of the mesencephalon (Text-fig. 3).
In the anterior part of the diencephalon, short, scattered fibers are seen. These
are oriented radially or circumferentially. In the posterior diencephalon some
longer fibers are seen and they are more densely distributed. Many of the fibers
in the lateral part of the posterior diencephalon are oriented in an anterior and
dorsal to posterior and ventral direction. Similarly oriented fibers are seen in
the anterior mesencephalon, constituting the posterior part of this area of
Initial neuroblast differentiation
505
differentiation. Neuroblast cells are oriented so that processes extend laterally,
ventrally, or from those in the ventral diencephalon and mesencephalon,
posteriorly, or occasionally anteriorly (Text-fig. 3). In some cases the processes
extend ventrally and then turn posteriorly.
The number of longitudinal fibers in the ventral diencephalon and mesencephalon becomes greater and the area in which they are found increases in size.
By the 18-somite stage, longitudinal fibers are present from the area just
posterior to the base of the optic stalk to the middle of the mesencephalon
Text-fig. 4. Group D: 21-somite embryo, sagittal sections. Diagram of the pattern
of neuroblast cells and nerve fibers in the diencephalon and mesencephalon. The
positions and orientation of representative cells and fibers are indicated; not all of
the cells and fibers actually visible in the embryo are shown (see Plate 1, figs. C,
D). O, Location of optic stalk. Arrows indicate boundaries of telencephalon (71),
diencephalon (£>) and mesencephalon (M). x 40.
(Text-fig. 3). There are also circumferential fibers at this level in the diencephalon and a few in the ventral mesencephalon. The longitudinal fibers appear to
be processes of longitudinally oriented neuroblasts in the ventral diencephalon
and mesencephalon and of neuroblasts whose processes extend ventrally and
turn at this level.
Nerve fibers appear in the dorsal mesencephalon by the 18-somite stage. At
this time the area covered by these fibers is discrete from the first area of differentiation. In the embryo in Text-fig. 3, segments of fibers can be seen scattered
along and to each side of the midline from the posterior boundary of the
diencephalon to the anterior end of the rhombencephalon.
D. 19- through 22-somite stage
The most striking feature of this period is the elaboration of the fiber pattern
within the lateral area of differentiation. Neuroblasts and processes become
considerably more numerous, and the segments of fibers visible in each section
are longer. In addition, a new center of differentiation develops just posterior
and ventral to the base of the optic stalk.
The lateral area of differentiation continues to expand. By the 21-somite
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K. M. LYSER
stage (Text-fig. 4) it extends from just posterior to the optic stalk into the
anterior part of the mesencephalon. At the anterior edge of the mesencephalon,
in the synencephalon (approximately the posterior third of the diencephalon,
which is demarcated for a time in the embryo from the anterior portion of the
diencephalon, or parencephalon, by a transitory constriction) and in the posterior parencephalon, there are circumferential fibers from the dorsal part of
the lateral wall to the level of the longitudinal fibers. Anterior to this they are
not found as far dorsally; just posterior to the optic stalk circumferential fibers
can be found from approximately the middle of the diencephalon to the level
of the longitudinal fibers. The most dorsal fibers in the posterior diencephalon
and anterior mesencephalon have a dorsal-ventral orientation, axons extending
more or less directly ventrally from the cell bodies (Plate 1, figs. C, D). Farther
ventrally some are at an angle from anterior-dorsal to posterior-ventral and
some from posterior-dorsal to anterior-ventral. Most of the fibers in the
anterior part of the parencephelon are oriented obliquely from anterior-dorsal
to posterior-ventral.
The ventral longitudinal fibers begin to form a more clearly outlined fasciculus. At the 21-somite stage they are seen from the area posterior to the optic
stalk to the posterior border of the mesencephalon, where they merge with
the longitudinal fibers of the hind brain. In the posterior parencephalon and
in the synencephalon the fasciculus is quite wide, extending from the middle
of the diencephalon to the ventral part of the lateral wall. The more dorsal
longitudinal fibers curve ventrally as they approach the mesencephalon where
they form a narrower fasciculus. There are fewer fibers in the fasciculus in the
mesencephalon than in the posterior part of the diencephalon; in the embryo
represented in the diagram (Text-fig. 4) the number decreases to about five at
the posterior end of the mesencephalon. In the anterior part of the parencephalon
the dorsal edge of the fasciculus also curves ventrally and the fibers decrease
in number until there are just a few longitudinal fibers at the level about one
fourth of the distance from the floor plate of the diencephalon to the roof.
PLATE 1
Fig. A. 16-somite embryo, group A. Ventral part of the posterior diencephalon, sagittal
section. A neuroblast cell with a process extending ventrally (AT) and two segments of fibers
(F) can be seen, x 2000.
Fig. B. 16-somite embryo, group B. Lateral diencephalon, dorsal to the middle, sagittal
section. The neuroblast (A0 has a process extending ventrally and slightly posteriorly along
the border between the nuclear and peripheral zones, x 2000.
Fig. C. 21-somite embryo, group D. The same embryo as in Text-fig. 4, mesencephalon
and diencephalon, sagittal section. Fibers and neuroblasts in the lateral diencephalon (D)
and longitudinal fibers in the ventral mesencephalon (M) can be seen in this section, x 240.
Fig. D. 21-somite embryo, group D. The same section as fig. C. Neuroblasts in the posterior
diencephalon with processes extending ventrally and obliquely can be seen, x 1000.
J. Embryol. exp. Morph., Vol. 16, Part 3
K. M. LYSER
PLATE 1
facing p. 506
/. Embryol. exp. Morph., Vol. 16, Part 3
PLATE 2
facing p. 507
Initial neuroblast differentiation
507
Cells seen at the level of the longitudinal fasciculus have processes extending
ventrally, at an angle anteriorly or posteriorly, or directly anteriorly or posteriorly. Sometimes two adjacent cells have processes extending in different
directions; in a few instances the processes can be seen to cross near the cell
bodies.
At the level of the optic stalk is another group of longitudinal fibers. These
are oriented at an angle from anterior-ventral to posterior-dorsal. No fibers
are visible between this group and the first longitudinal fibers and none can be
seen crossing the mid line. No neuroblast cell bodies have been seen in this area
in the embryos examined; it is not clear where the neuroblasts which give rise
to these fibers are located.
The fibers in the dorsal mesencephalon have a pattern similar to that at the
previous stage; they do not extend much farther ventrally during this period.
In the embryo illustrated (Text-fig. 4), a few short pieces of fibers are visible
on each side of the mid line.
E. 23- through 30-somite stage
During this period the areas of axon outgrowth which have appeared during
the previous stages are enlarged and neuroblasts of the oculomotor nucleus
begin to send out processes.
In the lateral diencephalon more neuroblasts send out processes ventrally or
obliquely, but their distribution does not change markedly (Text-figs. 5, 6;
Plate 2, figs. A, B). At the end of this period (Text-fig. 6, 28-somite embryo)
circumferential fibers can be seen throughout the lateral wall of the posterior
diencephalon. In the anterior diencephalon they are quite numerous in the ventral part of the lateral area, but much more sparsely distributed dorsally. At the
dorsal edge of the lateral fiber area in the synencephalon and posterior part of
the parencephalon there are a few fibers oriented longitudinally. There are also
a few which cross the posterior part of the synencephalon at an angle from dorsal
and posterior to anterior and ventral; that is, they seem to run from the dorsal
longitudinal fibers to the longitudinal fasciculus. At the anterior end of this
area, posterior to the optic stalk and just above the center of the lateral wall,
PLATE 2
Fig. A. 28-somite embryo, group E. The same embryo as in Text-fig. 6, lateral part of the
posterior diencephalon, sagittal section. This section shows cells and fibers at the middle of
the lateral wall, including oblique fibers which cross, x 800.
Fig. B. 28-somite embryo, group E. This is the next section medial to that in fig. A, showing
more ventrally located cells and fibers in the diencephalon (D) and mesencephalon (M),
including longitudinal fibers, x 800.
Fig. C. 28-somite embryo, group E. The same embryo as figs. A and B, mesencephalon,
sagittal section. This section is at the level of the oculomotor nerve. Its fibers can be seen
emerging from and outside of the neural tube. Sections of longitudinal fibers (L) can be seen
posterior to the oculomotor nerve, x 1000.
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K. M. LYSER
there are also a few fibers oriented longitudinally. At the posterior end of the
synencephalon fibers oriented more or less transversely seem to cross the
dorsal mid line. These fibers are not separated anteriorly and ventrally from
those of the lateral area or posteriorly from the fibers of the dorsal mesencephalon. Neuroblasts that have processes extending ventrally or obliquely can be
seen throughout the lateral diencephalon in sagittal sections; in transverse
sections, cells with processes extending laterally are apparent.
Fig. 5
Fig. 6
Text-fig. 5. Group E: 24-somite embryo, sagittal sections. Diagram of the pattern
of neuroblast cells and nerve fibers in the diencephalon and mesencephalon.
O, Location of optic stalk. Arrows indicate boundaries of telencephalon (71), diencephalon (D) and mesencephalon (M). x 40.
Text-fig. 6. Group E: 28-somite embryo, sagittal sections. Diagram of the pattern
of neuroblast cells and nerve fibers in the diencephalon and mesencephalon.
O, Location of optic stalk. Arrows indicate boundaries of telencephalon (T), diencephalon (D) and mesencephalon (M). x 40.
The number of fibers in the juxta-optic group increases also. In 24-somite
embryos (Text-fig. 5) more fibers than at the previous stages can be seen on
both sides posterior and ventral to the base of the optic stalk. As before, they
are oriented predominantly in an anterior and ventral to posterior and dorsal
direction. However, they are no longer separate from fibers of other areas.
Circumferentially oriented fibers are found as far anterior as the level of the
juxta-optic fibers, just dorsal to them. Posteriorly, the juxta-optic fibers merge
with the longitudinal fasciculus. In this embryo fibers cannot be seen crossing
the ventral mid line. By the 28-somite stage (Text-fig. 6) the fibers are more
numerous still, especially just posterior to the optic stalk, and there are some
fibers ventral and medial to it. In transverse sections, fibers crossing the mid
line at the level of the posterior edge of the optic stalk can be seen.
Initial neuroblast differentiation
509
At the 24-somite stage, the area of differentiation in the mesencephalon is
a little more extensive than in embryos of the 19- through 22-somite group.
In addition to circumferential fibers at the anterior edge of the mesencephalon,
there are now fibers in the ventral half of the lateral wall as far posterior as
the middle of the mesencephalon. In this area some neuroblasts with fibers
extending ventrally or obliquely posteriorly and ventrally can be seen. As before,
there are circumferential fibers in the dorsal midbrain. Occasionally a fairly
long fiber extends ventrally to the middle of the mesencephalon. In the dorsal
part there are also a few longitudinal fibers.
By the end of this period (Text-fig. 6), circumferentially oriented fibers are
present throughout the lateral wall of the midbrain. They form one continuous group, which also includes transversely oriented fibers in the mid-dorsal
region. Most of the lateral fibers are dorsal-ventral or slightly oblique. Another
new feature which appears during this period is longitudinally oriented fibers
at the middle of the lateral area of the mesencephalon. The ventral ends of some
of the circumferential fibers can be seen to turn posteriorly at this level, thus
contributing to the longitudinal group. Neuroblasts with processes extending
ventrally or obliquely can be seen in the mesencephalon.
The number of fibers in the ventral longitudinal fasciculus and the number
of cells with processes located at this level also continue to increase. In the
younger embryos of this group (Text-fig. 5), there are only a few longitudinal
fibers in the posterior mesencephalon and not many in the anterior diencephalon.
By the 28-somite stage (Text-fig. 6), the ventral longitudinal fasciculus contains
a considerable number of fibers throughout. The fibers are more or less parallel
to each other, though there is some crossing. In the mesencephalon the fasciculus
is located at the ventral-lateral corner of the neural tube. In the synencephalon
and posterior parencephalon it is in a more dorsal position and is wider; the
most dorsal fibers are at about the middle of the diencephalon. Anterior to this
the fasciculus becomes narrower and is continuous with the fibers of the juxtaoptic area. There are circumferential fibers at the level of the longitudinal
fasciculus, that is, crossing the longitudinal fibers, particularly in the posterior
diencephalon. A few circumferential fibers which turn anteriorly or posteriorly
at the level of the longitudinal fibers are visible. Neuroblasts with processes
extending posteriorly, or occasionally anteriorly, posteriorly and ventrally or
anteriorly and ventrally, can be seen among the longitudinal fibers.
Fibers of the oculomotor nerve are first recognizable in 25-somite embryos.
They are located near the anterior end of the mesencephalon at the medial edge
of the longitudinal fasciculus. The oculomotor area is not completely separate
from other areas of differentiation, since by this time neuroblasts are recognizable at the level of the longitudinal fasciculus, just lateral to the oculomotor
area. No specific pattern of arrangement within the oculomotor area is discernible during the period under consideration. In the youngest embryos in
which oculomotor fibers can be identified, a few can be seen emerging from or
32
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510
K. M. LYSER
just outside the ventral mesencephalon on each side. For example, in one
25-somite embryo, sectioned transversely, all the oculomotor fibers can be seen
in four sections (six sections distant from the anterior edge of the mesencephalon
and eighteen sections from the posterior edge). They are located at the medial edge
of the longitudinal fasciculus. In the first section there are three fibers ventral
to but not touching the neural tube on the right side and one on the left which
is touching the edge of the neural tube but cannot clearly be seen to come from
within the mesencephalon. In the second section there are two fibers emerging
from the mesencephalon on the right and one on the left. In the third section
there is one fiber emerging on the right. In the fourth section there is one fiber
on the right which is touching the edge of the mesencephalon but not definitely
emerging in this section. The more lateral emerging fiber on the right in the
second section turns out of the section just inside the edge of the neural tube.
The other emerging fibers seem to come from within the nuclear zone in the
same section; the cell bodies cannot be identified. Just anterior to the fibers of
the oculomotor nerve there are some radial fibers medial to or at the level of the
longitudinal fibers which may also be oculomotor. Some are in the nuclear zone,
some pass from the nuclear zone into the peripheral zone.
In slightly older embryos, more fibers and some neuroblasts of the oculomotor nucleus can be seen anterior and posterior to the first fibers as well as
among them (Text-fig. 6; Plate 2, fig. C). They are located at the medial part
of the longitudinal fasciculus and just medial to it. A few of the neuroblasts
which give rise to the oculomotor fibers can be identified near the edge of the
neural tube at the medial side of or just medial to the longitudinal fasciculus.
They have processes that extend ventrally and emerge from the mesencephalon.
In embryos sectioned sagitally, neuroblasts can be seen which have processes
that extend posteriorly and then turn ventrally to leave the neural tube.
In summary, during the period of initial neuroblast differentiation in the
diencephalon and mesencephalon the following sequence has been observed.
The first neuroblasts with processes were seen in 14-somite embryos in the
lateral part of the posterior diencephalon. Their axons extend laterally or
ventrally from the cell bodies; the lateral fibers turn ventrally. By the 15- or
16-somite stage the beginning of a ventrally located longitudinal group of fibers
is indicated. The longitudinal fibers are apparently processes of laterally located
cells which turn posteriorly at this level and of cells located among the longitudinal fibers. At about the 18-somite stage a new area of differentiation appears
in the dorsal mesencephalon with fibers extending circumferentially. The next
area of differentiation is visible by the 21-somite stage in the juxta-optic region.
All of these areas are enlarged so that by the 30-somite stage neuroblasts
and fibers are present, fairly evenly distributed, over most of the lateral wall of
the diencephalon and mesencephalon. The more dorsal and lateral fibers are
mainly circumferential or oblique. A prominent group of ventrally located
longitudinal fibers extends from the juxta-optic area through the mesencephalon,
Initial neuroblast differentiation
511
and some longitudinal fibers are also seen dorsally in the posterior diencephalon
and along the middle of the lateral wall of the mesencephalon. Oculomotor
fibers, from neuroblasts in the ventral mesencephalon, are first recognizable
at about 25 somites.
DISCUSSION
The general pattern of differentiation as described above agrees for the main
part with previous studies which include early stages of nerve-fiber development
(Tello, 1923; Windle & Austin, 1936; Van Campenhout, 1937). Observation of
fibers in slightly younger embryos in the present study may be due to differences
in staining procedures, as well as possible variations in embryos and determination of stages. The present study is concerned primarily with details of development during this period and does not include study of older stages which would
be necessary for more extensive discussion of the identity of neuroblasts and
fiber groups in terms of adult nuclei and tracts.
From these observations of the development of the diencephalon and mesencephalon in 13- to 30-somite chick embryos, the following generalizations can
be made about the way in which the initial phase of differentiation proceeds.
(1) Several different areas of differentiation can be recognized in the diencephalon and mesencephalon, which appear in the embryo in regular sequence.
(2) In each area the first neuroblasts are distributed in a scattered fashion, the
area is progressively enlarged by the differentiation of additional cells at its
edges, and at the same time new cells differentiate within the old area. (3) Processes of the cells in each area, or part of an area, grow out in a generally
consistent and characteristic direction, with small irregularities and variations
of the courses being typical.
The sequence of initial neuroblast differentiation in the diencephalon and
mesencephalon is more complex than the anterior-posterior, dorsal-ventral
pattern seen in the development of the embryo in general. Differentiation
begins in the diencephalon and mesencephalon after it has started farther
posteriorly, in the hind brain. Within the mid- and forebrain it occurs first in
the lateral wall of the posterior diencephalon and anterior mesencephalon,
followed by the more posterior and dorsally located dorsal mesencephalon, and
then by the more anterior juxta-optic area. Furthermore, in each area, differentiation spreads from the initial location in various directions. Such a characteristic pattern implies a specific regional differentiation within the neural tube
at the time of initial differentiation of neuroblasts. This organization may result,
directly or indirectly, from the very early regional differentiation of the medullary plate and neural tube. The latter is demonstrated by the distinctive gross
form of various parts of the brain and by developmental capacities under
experimental conditions, such as experimental regional induction or development of isolated regions, including development of characteristic patterns of
function (Corner, 1964). Separation of parts of the neural tube in vivo during
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512
K. M. LYSER
initial neuroblast differentiation demonstrates the ability of these regions to
differentiate further, morphologically and functionally, without continuity with
one another (Rhines & Windle, 1944; Hamburger & Balaban, 1963; Hamburger,
Balaban, Oppenheim & Wenger, 1965). This does not necessarily imply complete independence of differentiation in the neural tube. There is some evidence
from organ-culture studies that differentiation in the central nervous system is
influenced by the surrounding environment, including adjacent tissues (Szepsenwol, 1940a, b; Lyser, 1966). Though certain adjacent tissues affect histological
organization in the spinal cord (Holtfreter, 1939; Holtfreter & Hamburger,
1955), the effect could be somewhat non-specific in terms of cellular differentiation, comparable to certain cases of mesenchymal-epithelial interactions
(Grobstein, 1953, 1962; McLoughlin, 1961). The influencing factor could
support development of a cell type determined by the specificity of the region
of the neural tube.
The characteristic, specific pattern observed pertains to the location and
sequence of differentiation of groups of neuroblasts. No evidence of a specific
pattern of individual cells has been found. This possibility cannot be ruled out by
observations such as those in this work, where all the cells cannot be seen
because of their orientation relative to the plane of section, but it seems unlikely that such would occur.
The scattered distribution of the first cells within an area can probably be
correlated with their early differentiation. As regards each individual neuroblast
cell, the sequence of proliferation followed by differentiation, and especially
the absence of division once morphological differentiation has begun, is adhered
to. Since proliferating cells are distributed all along the neural tube, and since
much proliferation is still to take place in all areas, it is not surprising that
scattered individual cells from among the neural epithelial cells differentiate
first. This leaves the rest to divide until a sufficient number have been produced
to form a mantle layer and an inner neural epithelial layer.
What is responsible for the initiation of differentiation in certain cells is
a question still to be answered. The explanation must cover the localization
of the process in specific areas and also selection of a limited number of cells
in each area out of a large population, many of which would presumably be
able to differentiate in the same way.
The sequence of differentiation in the earliest neuroblasts appears to be
different from that of the classical description, applicable to later stages; but
it is consistent with the description of Windle & Austin (1936; see Windle &
Baxter, 1936, and Lyser, 1964). The presence of radial cells, circumferential
cells and cells with an orientation intermediate between these suggests that the
neuroblasts begin to send out processes while still having the position and orientation of neural epithelial cells. They subsequently shift to a position where the
mantle layer will form and sometimes, as in the lateral diencephalon, change
their orientation.
Initial neuroblast differentiation
513
The characteristic pattern of development pertains to the orientation of
neuroblast cells and the courses of their axons as well as to the location and
sequence of differentiation. Radial orientation of cell bodies is not necessarily
included in this category, but rather may be a reflexion of the sequence of
differentiation in the early neuroblasts. Otherwise, cell bodies generally have
fairly consistent orientations which can be considered characteristic of each
area. The courses of individual fibers suggest a specificity in regard to the general
direction, with small variations probably due to a number of other factors
influencing the pathway at the same time. Small deviations in the courses of
individual fibers suggest that they are following a path of least mechanical
resistance around various obstacles. Mechanical factors have been shown to be
important in determining the pathways of nerve fibers under various experimental conditions, and could exert some effect on an outgrowing fiber in the
embryo also. For example, nerve fibers need a surface or interphase along which
to grow (Lewis & Lewis, 1912; Harrison, 1914). Nerve processes grow along
the fibrils in a plasma clot that have been oriented by stroking (Weiss, 1934).
In regenerating tadpole tails, nerve fibers grow along the surface of fibroblasts
and of other nerve fibers and also can be blocked by fibroblast cells and processes when these structures form obstructions in the paths of the fibers (Speidel,
1933).
However, it seems that the overall pattern would be much more random,
instead of fairly consistently the same for the fibers in a given area, if there
were not some more specific factor directing the fiber in a particular direction.
No explanation of such a directive mechanism is apparent, but initial outgrowth of fibers may be comparable to experimental situations, including
regeneration of fibers, where there is evidence of very specific selection of pathways. For example, if the hind brain is reversed or if an obstruction is placed
in its path, the axon of Mauthner's cell eventually assumes a position in the
cord at, or fairly near, its normal location (Piatt, 1943, 1947; Stefanelli, 1950,
1951). Also, after removal of spinal ganglia in tadpoles previous to the development of the hind limb there is an almost normal motor pattern and there are
no nerves in the sensory pathways. After removal of ventral horn cells, a normal
sensory pattern develops (Taylor, 1943, 1944). The most striking example of
the growth of particular fibers to particular end structures is the regeneration
of amphibian and teleost optic fibers from each part of the retina to a specific
area of the optic tectum, even after the eyeball is rotated (Sperry, 1945, 1948).
Since a consistent pattern of bifurcating fibers is not apparent, in the diencephalon and mesencephalon the branching of axons observed once in a while
may represent temporary branches from fine processes of the growth cone, one
of which will be established as the next segment of the fiber and the others
withdrawn. The absence of many random fibers argues against formation and
retraction of any but very short branches. Selection from among fibers reaching
various other cells, as seen, for example, in regenerating tadpole tails, where
514
K. M. LYSER
cutaneous fibers sometimes grow toward muscles instead of toward the surface
and are eliminated by retraction or degeneration (Speidel, 1942), is unlikely
to occur here.
The extent of cellular differentiation, or determination, with respect to neuron
type at the time of initial outgrowth of axons, is not known. The morphological
pattern suggests differentiation at least of cells in one major area of development
as distinct from those in another, or as embarked on a different course of
development. The neuroblasts in the lateral diencephalon area, with processes
extending ventrally and posteriorly, are distinct from those of the oculomotor
nucleus, with processes passing out of the neural tube. However, within each
area cells may or may not be differentiated into more specific types, which will
later be included in various nuclei or parts of nuclei and have characteristic
connexions. One of the simpler examples of differentiation within a group of
neuroblast cells related to the present observations is the oculomotor nucleus.
There is no indication of separate groups of cells at the beginning of differentiation, though the cells innervate four different extrinsic eye muscles, and hence
are four functionally different groups of neurons, which are probably arranged
in a particular way in the adult. (This has not been demonstrated in birds but
there is some information on functional localization in mammals; see Warwick,
1953).
Regardless of the extent of specificity present in the neuroblasts at the time
they begin to send out processes, the pattern of the location and the sequence
of initiation of differentiation does not parallel that of future nuclei. For
example, the first neuroblasts in the diencephalon-mesencephalon area seem
to be scattered so as to include thalamo-tegmental and thalamo-bulbar cells
as well as those of the future nucleus of the medial longitudinal fasciculus in
one continuous area. This pattern suggests that the control of the initiation of
differentiation may be separate from factors determining particular pathways
and connexions specific for each type of neuron; cells are somehow 'triggered'
to differentiate but the particular type of differentiation depends on some
mechanism already set within the cells, or on other factors influencing it at the
same time.
The pattern of differentiation of the first neuroblast cells and their processes
in the diencephalon and mesencephalon of the chick embryo thus points out
several problems: What is responsible for the initiation of differentiation in
specific locations? How is the specificity of each individual cell acquired? What
is the mechanism of directional growth of a nerve fiber? Does the pattern of
initial differentiation influence subsequent formation of nuclei? It is hoped that
this study of the pattern in the normal embryo will be the basis of further
investigations to obtain information on some of these questions.
Initial neuroblast differentiation
515
SUMMARY
1. The pattern of cells and nerve fibers in the diencephalon and mesencephalon during the initial stages of neuroblast differentiation has been studied
in silver-impregnated sections of 13- through 30-somite chick embryos.
2. The first neuroblasts were seen at the 14-somite stage, located in the
lateral part of the posterior diencephalon with axons extending laterally and
ventrally. Ventral longitudinal fibers appear by the 15- or 16-somite stage.
Centers of differentiation appear subsequently in the dorsal mesencephalon,
and the juxta-optic area. Oculomotor fibers appear at about the 25-somite stage.
3. The differentiating neuroblasts are scattered; the initial areas are extended
by differentiation of additional neuroblasts among the first cells and at the
edges of original areas.
4. The orientation of neuroblast cell bodies and the directions of fibers are
characteristic for each area.
5. These observations demonstrate a specific pattern of development within
the nervous system and emphasize the need for further investigation of the factors
controlling the various aspects of differentiation.
RESUME
Le developpement du diencephale et du mesencephale d'embryon de poulet
au cours des phases initiales de differentiation des neuroblastes
1. On a etudie la disposition des cellules et des fibres nerveuses du diencephale et du mesencephale au cours des stades initiaux de la differenciation des
neuroblastes sur des coupes d'embryons de poulet de 13 a 30 somites, impregnees a 1'argent.
2. Les premiers neuroblastes ont ete observes au stade 14 somites, localises
dans la partie laterale du diencephale posterieur avec des axones s'etendant
lateralement et ventralement. Des fibres longitudinales ventrales apparaissent
aux stades 15 ou 16 somites. Des centres de differentiations apparaissent par
la suite dans le mesencephale dorsal et la region juxta-optique. Les fibres
oculomotrices apparaissent aux environs du stade 25 somites.
3. Les neuroblastes en differenciation sont disperses; les zones initiales
s'etendent par differenciation de neuroblastes additionnels au milieu des
premieres cellules et sur les bords des zones d'origine.
4. L'orientation des corps cellulaires des neuroblastes et la direction des
fibres sont caracteristiques pour chaque zone.
5. Ces observations mettent en evidence un plan de developpement specifique
dans le systeme nerveux et soulignent la necessite de nouvelles recherches sur les
facteurs qui controlent les divers aspects de la differenciation.
The author wishes to express her appreciation to Professor Leigh Hoadley for all of his
help in many ways. Part of this work was done during the tenure of a National Science
Foundation Pre-doctoral Fellowship.
516
K. M. LYSER
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