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Experimental data on the histogenesis of ganglion cells in the sympathetic trunk of the chick.

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EXPERIMENTAL DATA ON T H E HISTOGENESIS
O F GANGLION CELLS I N T H E SYMPATHETIC
TRUNK O F THE CHICK
HARRY E. RAYBUCK
Anatomical Laboratories of Saint Louis University, Saint Louis, Missouri, and
the Medical College of Georgia, Augusta, Georgia
ELEVEN FIGURES
There are at present three schools of thought relative to
the origin of the autonomic ganglion cells. According to one
school these cells are derived exclusively from the neural
crest ; another derives them locally from the mesenchyme. According to the third they are derived from the neural tube.
The pertinent literature relative to the histogenesis of the
autonomic ganglia has been reviewed repeatedly. Among the
more recent reviews may be mentioned those of Yntema and
Hammond ( '47) and Kuntz ('53). The present investigation
was undertaken to obtain by experimental methods, more decisive data relative to the origin of the ganglion cells in the
sympathetic trunks.
MATERIAL8 AND METHODS
Chick embryos were chosen as the experimental materiai.
Operative procedures were carried out at approximately 4548 hours of incubation (16-18 somite stage). Determination
of age was facilitated by the excellent chart of Hamburger
and Hamilton ( '51). The operation consisted of destroying
the neural crest or the neural crest and the dorsal portion of
the neural tube unilaterally or bilaterally throughout a series
of successive segments. Destruction of the tissue was accomplished by applying a heated sharp needle along the dorsal
midline of the embryo in the selected region.
603
604
HARRY E. RAYBUCI<
The eggs were incubated with the air sac uppermost. By
the use of an electric drill an opening approximately 1em
square was made in the shell and the shell membrane over the
air sac. The vitelline membrane was removed with fine
needles. The embryo was stained by placing blocks of agar
impregnated with a 0.5% solution of rhodamine B in contact
with it for approximately one minute. A t completion of the
operation the egg shell was sealed with adhesive or transparent plastic tape and returned to the incubator. Aseptic
technique was practiced throughout the operation as f a r as
possible. Post operative incubation varied from 1 to 9 days.
At the termintaion of incubation the embryo was removed
and fixed in formalin, Bouin’s fluid, or a solution of equal
parts of pyridine and water. Serial sections were cut at thicknesses of 12 to 15 p and stained with hematoxylin and eosin,
Holmes’ silver nitrate, toluidine blue, or a modified Cajad
silver stain. Only embryos in which the neural crest was
completely destroyed in 4 or more successive segments were
used. Twelve embryos in the present series were found to be
satisfactory for study.
OBSEEVATIONS
A . Normal embryos
I n embryos fixed early in the third day of incubation, approximately 50-55 hours, displacement of cells from the neural
tube along the ventral nerve roots may be observed. The cells
being displaced are similar in appearance to cells in the wall
of the neural tube. Their derivation from the mantle layer of
the neural tube is apparent. During the third day of incubation the neural crests are displaced ventrad and become
segmented to form the dorsal root ganglia. Displacement of
cells of neural crest origin along the dorsal nerve roots is
also apparent. The migration of cells from the neural tube
along the ventral nerve roots is usually concluded at 92-98
hours of incubation, whereas migration along the dorsal nerve
root continues to approximately 100 hours of incubation. Both
HISTOGENESIS OF SYMPATHETIC GANGLIA
605
the primary and the secondary sympathetic trunks comprise
cells that are displaced along nerve roots.
During the crucial third day of incubation the neural crests
assume a position adjacent to the ventral nerve roots. It
becomes impossible, therefore, to identify the cells of neural
tube origin and those of neural crest origin distal to the union
of the ventral and dorsal nerve roots. Elimination of the cells
of neural crest origin, therefore, is advantageous in attempts
to determine whether sympathetic ganglion cells are derived
from the neural tube.
B. Ezperimental embryos
For descriptive purposes the operated embryos may be divided into two categories: (1) those with the neural crest
and the dorsal portion of the neural tube destroyed in a series
of successive segments and (2) those with complete destruction of the neural crests and the neural tube in a series of
successive segments.
I n the first category the neural crest or the neural tube
were destroyed either unilaterally or bilaterally in varying
numbers of successive segments (figs. 1 and 2). Destruction
of the neural tube varied in degree. The actual extent of the
damage could only be determined after serial sections of the
embryos were made. The experimental embryos in which
spinal ganglia were absent in less than 4 successive segments
were not used in this study. The number of segments devoid
of neural crest derivatives was determined in the following
manner. Sections were counted from the middle of one spinal
ganglion to the middle of the next, if the damage was unilateral, or from the beginning of one intervertebral foramen to
the beginning of the next if the damage was bilateral. Counts
of the same kind were also made in unoperated chicks of the
same age at the same level and cut at the same thickness.
This afforded a reasonably accurate estimate of the number
of sections in a segment. On the basis of this criterion, 1 2
embryos were found to be suitable for study.
606
HARRY E. RAYBUCK
The average extent of neural crest destruction was 5 to 6
successive segments. In some embryos the neural crests were
destroyed in 9 successive segments. I n embryos devoid of
spinal ganglia and dorsal nerve roots in a series of successive
segments, but with ventral nerve roots, sympathetic primordia
could always be found in these segments (figs. 1, 2, and 3 ) .
When the destruction was unilateral there was usually a contrast between the operated and the unoperated side. The sympathetic primordia were smaller on the side devoid of neural
crest derivatives than on the opposite side. I n embryos in
which the destruction was bilateral the sympathetic primordia
were usually of approximately equal size on both sides but
somewhat smaller than those in the same segments of normal
embryos.
I n embryos fixed 24 hours after the operation the operated
segments usually were devoid of sympathetic primordia.
Early primordia could be seen in some instances and cells of
neural origin were present in the ventral nerve roots. The
late appearance of sympathetic primordia undoubtedly is due
to the trauma of the operation. This, according to Jones
('37), affords an explanation of the failure of Miiller and
Ingvar ('23) to find sympathetic primordia in some of their
operated embryos.
I n some embryos the destruction of the neural tube was so
extensive that in some segments only a small ventral nerve
root was present and some Segments were devoid of ventral
nerve roots. When the ventral nerve roots were reduced in
size the sympathetic primordia were correspondingly reduced. Complete absence of ventral nerve roots was associated with the absence of sympathetic primordia in some segments. In some instances sympathetic primordia were present
in segments devoid of ventral nerve roots, probably due to
longitudinal displacement of cells from the adjacent segment
in which a ventral nerve root and sympathetic primordium
were present.
When more than the dorsal portion of the neural tube was
destroyed sympathetic primordia showed a corresponding
HISTOGENESIS O F S Y M P A T H E T I C GANGLIA
607
reduction in size. The visceral rami were also reduced. The
role of the preganglionic center in the neural tube in the formation of the sympathetic primordia is therefore apparent.
The most critical part of this study is concerned with the
nature of the cells in the sympathetic primordia in segments
in which spinal ganglia and dorsal nerve roots are absent.
In the embryos fixed after 4 to 6 days of incubation following
the operation, the differentiation of most of the nerve cells
in the Sympathetic primordia has not advanced beyond the
neuroblast stage. I n the more rostra1 thoracic segments of
somc of the embryos fixed 6 days after operation some cells
could be recognized as neuroblasts (fig. 4). These cells usually
were oval in outline, although the beginning of process formation could be observed (fig. 5 ) . Their nuclei were spherical
and the chromatin stained darkly. Some of them exhibited
dispersed chromidial substance. In general the neuroblasts
stain more intensely than the more elongated supporting cells
and can be readily identified.
I n the embryos fixed after 7 to 9 days of incubation after
operation some cells in the sympathetic primordia have differentiated beyond the neuroblast stage and are easily recognized as multipolar neurons. I n some of the embryos fixed
8 and 9 days after operation neural crest derivative were
absent in 7 successive segments. In one of them 9 successive
segments were devoid of spinal ganglia and dorsal nerve
roots. I n this embryo a well developed sympathetic trunk,
somewhat smaller than normal, was present throughout the
entire series of segments affected by the operation. Many of
the cells in the sympathetic trunk ganglia in all these segments
had differentiated into bipolar neuroblasts and multipolar
neurons. The multipolar neurons exhibited spheroid dark
staining nuclei and dendritic processes. The cytoplasm also
included dark staining chromidial substance (figs. 6 and 7).
These multipolar cells undoubtedly represent sympathetic
ganglion cells. I n the absence of neural crest derivatives in so
608
HAFiRY E. RAYBUCK
many successive segments, they can be interpreted only as
ganglion cells of neural tube origin.
The differentiation of neuroblasts into more advanced
stages, beginning in the thoracic region, could be observed
in embryos incubated 8 and 9 days after operation. Cells in
the multipolar phase could be found throughout the sympathetic trunk. The cells in the multipolar phase are best demonstrated in areas where there is a paucity of ganglion cells.
I n such areas the processes of the cells are most apparent.
The most significant finding in this study is the demonstration of ganglion cells in the multipolar phase in the middle
segments of a series of 6 or more successive ones that were
devoid of spinal ganglia and dorsal nerve roots (figs. 8 and
9). Such extensive damage excludes the possibility of longitudinal displacement of cells of neural crest origin from segments more rostral in which neural crest derivatives were
present into the segments in question. The multipolar neurons
in the sympathetic primordia in these segments, consequently,
must be of neural tube origin.
In embryos in which the neural crest was destroyed unilaterally the sympathetic primordia on the operated side were
compared with those on the opposite side. In every instance
those on both sides presented the same histological appearance. Except for a reduction in the size of the sympathetic
trunk ganglia there was no apparent difference. The operation appeared to have no marked retarding effect on the
maturation of the ganglion cells (figs. 10 and 11).
I n 4 embryos the neural tube was completely destroyed in
4 to 6 successive segments. Except in the two most rostral
ones, the segments that were devoid of a neural tube were
also devoid of sympathetic primordia. Such findings indicate
that sympathetic primordia will not form in the absence of
the neural tube. The primordia present in the rostral segments of the affected area undoubtedly represent longitudinal
displacement caudad from the adjacent rostral segment in
which ventral nerve roots were present.
HISTOGENESIS O F SYMPATHETIC GANGLIA
609
DISCUSSION
The data obtained in this study are in complete agreement
with the earlier findings of Kuntz ( '20, '22)' Jones ('37) and
Brizzee ( '49). The experimental data demonstrate that the
sympathetic trunks may be formed and that multipolar ganglion cells may appear in them in the absence of spinal ganglia
and dorsal nerve roots. They substantiate the concept that
cells of medullary origin become displaced into the sympathetic trunks. They are not in accord with the conclusions
advanced by Miiller and Ingvar ( '23)' Van Campenhout ( '31)'
Yntema and Hammond ( '45)' Hammond and Yntema ( '47)'
and Hammond ( '48,'49) that the neural crest is the exclusive
source of sympathetic ganglion cells. Neither do they corroborate those of Tello ('25, '45) and others who have supported the point of view that the sympathetic primordia have
a mesodermal origin.
The longitudinal displacement of cells of neural crest origin
from adjacent unoperated segments into segments that are
devoid of spinal ganglia and dorsal nerve roots has been emphasized, especially by Hammond and Yntema ( '47)' to explain the results reported by other investigators that are incompatible with the point of view that all autonomic ganglion
cells are derived from the neural crests. This explanation
cannot be regarded as valid. Jones ('41) demonstrated rather
conclusively that maximal longitudinal displacement of cells
of neural origin from one segment to more caudal ones is
three segments and that there is no displacement from one
segment into a more rostra1 one. Rrizzee and Kuntz ('49)
reported displacement caudad through two se,gnents. The
maximum longitudinal displacement observed in the present
study was caudad through two segments. The displaced cells
formed an unsegmented cluster, whereas the normal sympathetic trunk primordia are segmental and connected with the
ventral nerve roots. There is no complete agreement relative
to the extent of longitudinal displacement of cells that may
take place along the sympathetic trunks. I n view of the data
available, neuroblasts or multipolar neurons in segments de-
610
HARRY E. RAYBUCK
void of neural crest derivatives that are separated from segments with neural crest derivatives by three or more intervening segments cannot be accounted for by longitudinal
displacement of neural crest cells. They obviously represent
cells that have been displaced from the neural tube along the
ventral nerve roots.
The demonstration of neuroblasts and multipolar neurons
in the sympathetic trunks in segments that are devoid of
neural crest derivatives proves that the neural tube is a
source of sympathetic ganglion cells. The present esperimental data do not preclude the possibility that cells of neural
crest origin may differentiate into sympathetic ganglion cells.
The displacement of cells of neural crest origin into the
sympathetic trunk primordia is generally conceded. I n the
light of Brizzee’s ( ’49) investigation it is possible to account
for these cells as supporting elements. It need not be assumed
that they differentiate into ganglion cells. The spinal ganglion cells, which are neural crest derivatives are afferent in
function, whereas, sympathetic ganglion cells, like the neurons
in the ventral portion of the neural tube, are efferent. It is
most logical to assume, therefore, that ganglion cells in the
sympathetic trunk are derived from the neural tube rather
than from the neural crest.
SUMMARY
The neural crest or the neural crest and varying amounts of
the dorsal portion of the neural tube have been destroyed
throughout a series of successive segments in chick embryos
of 45 to 48 hours of incubation. Incubation was continued
after operation from 1to 9 days. Primordia of the sympathetic trunks arose in all segments that were devoid of neural
crest derivatives in which ventral nerve roots were present.
Neuroblasts and multipolar neurons have been demonstrated in the spmpathetic trunks in segments devoid of spinal
ganglia and dorsal nerve roots f a r enough removed from
segments in which neural crest derivatives were present to
exclude the possibility of accounting for them on the basis
HISTOGENESIS OF SYMPATHETIC GANGLIA
611
of longitudinal displacement of neural crest cells. Such cells
must have their origin in the neural tube and bc displaced
along ventral nerve roots into the sympathetic primordia.
Thc data presented support the point of view that most, if
not all, of the cells that become differentiated into ganglion
cells in the sympathetic trunks arc derived from tho neural
tube.
LITERATURE CITED
BRIZZEE,K. R. 1949 Histogenesis of the supporting tissue in the’spinal m d
the sympathetic trunk ganglia in the chick. J. Comp. Neur., 91:
129-146.
BRIZZEE,
K. R., AND A. KUXTZ 1949 The histogenesie o f sympathetic ganglion
cells. J. Neuropath. and Exp. Neur., 9 : 164-171.
HANBCRGER,
v., AND H. HAMILTON1951 A series of normal stages in the development of the chick embryo. J. Morph., 83: 49-92.
HAXIYOBD,
W. 8. 1948 Removal of tho neural tube and its effect on the development of the sympathetic nervous system. Anat. Rec., 100: 6 i l .
1949 Formation of the sympathetic nervous system in tho trunk
of the chick embryo following removal of the thoracic neural tube.
J. Comp. Neur., 91: 67-85.
HAMMOSD,W. S., AND C. L. YNTENA 1947 Depletion of the thoracolumbar
sympathetic system following removal of the neural crest in the
chick. J. Comp. Neur., 86: 237-253.
JONES, D. 5. 1937 The origin of the sympathetic trunk in the chick embryo.
Anat. Ree., 70: 45-54.
1941 Further studies on the origin of sympathetic ganglia in the
chick embryo. Anat. Roc., 79: 7-15.
KUNTZ,
A. 1920 The development of the sympathetic nervous system in man.
J. Comp. Keur., 36: 173-229.
1923 Experimental studies on tho histogenesis of the s p p a t h e t i c
nervous system. J. a m p . h’eur., 34: 1-36.
1953 The Autonomic Nervous System. Lea and Fcbiger, Philadelphia. Chap. VI, 117-134.
1923 Ueber den Ursprung des Sympathicus beini
MIULLER,E., ASD S. INQVAR
IIiinchen. Arch. mikr. Anat., 99: 650-671.
TELLO, J. F. 1925 Sur la formation des chaines primaire et secondaim du
grand sympathique dans I’embryo di poulet. Trav. Lab. Biol., Univ.
Nadrid, 23: 1-28.
1945 Alpinas observaciones mas sobre las primeras fases del desarrola del simpatica en el pollo. Trab. del Inst. Capal Invest. Biol.,
87: 103-149.
VAN CAXPENHOUT,E. 1931 Le developmentc du systeme nerveux sympathique
chez le poulet. Arch. Biol., 46: 479-507.
---
612
HARRY E. RAYBUCK
1945 Depletions and abnormalities in
YNTEMA,C. L., AND W. S. HAMMOND
the cervical sympathetic system of the chick following extirpation of
the neural crest. J. Exp. Zool., 100: 237-263.
1947 The development of the Autonomic Nervous System. Biol.
Rev., 23: 344-359.
PLATE 1
EXPLANATION OF FIGURES
1 Photomicrograph demonstrating unilateral damage to the neural tube. The
dorsal root ganglion (D) is present on the left and absent on the right
side. A ventral root (V) and a sympathetic primordium (S) are present
on both sides. X 100.
2
Photomicrograph demonstrating bilateral damage to the neural tube. A
sympathetic primordium (S) is present on both sides; a ventral root ( V )
is present on the right side extending through the intervertebral foramen.
Spinal ganglia are absent. X 100.
3
Photomicrograph demonstrating the presence of a ventral root ( V ) and a
sympathetic primordium (S) in the absence of dorsal nerve root and ganglion. x 100.
H I S T O G E N E S I S OF S Y M P A T H E T I C GANGLIA
HARRY E. RAYBUCK
PLATE I
PLATE 2
EXPLANATION OF FIGURES
4 Photomicrograph of a sympathetic primordium in the middle segments of
a series of seven successive segments devoid of neural crest derivatives.
The majority of cells are in the neuroblast stage but some early process
formations may be observed. X 430.
5
Photomicrograph of a sympathetic primordium in the middle segments of
a series of eight successive segments devoid of neural crest derivatives.
The cells show early process formations and dispersion of chroinidial substance. x 1250.
6 Photomicrograph of a sympathetic primordium in the middle segments of
a series of nine successive segments free of neural crest derivatives. The
cells are in early multipolar phase and exhibit process formation. X 1250.
7
Photomicrograph of a sympathetic priniordium in the middle segments of
a series of nine successive segments free of neural crest derivatives. Note
the definite process formation and dispersion of chromidial substance in the
cytoplasm. X 1250.
8 Photomicrograph of a sympathetic primordium in the middle segments of
a series of seven successive segments devoid of neural crest derivatives.
The cells are in bipolar and multipolar phase.
x
1250.
9 Photomicrograph of a multipolar ganglion cell in a sympathetic primordium
in the middle segments of a series of eight successive segments devoid of
neural crest derivatives. X 1250.
G14
PLATE 2
H I S T Q G E N E S I S O F SYMPATHETIC GANGLIA
IIARRY E. RAYBUCP
615
PLATE 3
EXPLANATION O F FIGURES
10 Photomicrograph of a sympathetic primordium in the middle segments of
a series of eight successive segments devoid of neural crest derivatives.
Compare with figure 11. X 430.
11 Photomicrograph of the contra-lateral sympathetic primordium of the same
segment with neural crest derivatives present. This coniparison demonstrates
the usual difference in the operated and non-operated primordia. Note the
decrease i n cellularitr and size of the operated as coinpared to the control.
X 430.
fi 1 f i
HISTOGENESIS O F SYMPATHETIC GANGLIA
PLATE 3
HARRY E. RAYBUCK
617
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