/. Embryol. exp. Morph. Vol. 19, 2, pp. 109-19, April 1968
With 2 plates
Printed in Great Britain
109
Analysis of the development of the nervous system
of the zebrafish, Brachydanio rerio
I. The normal morphology and development of the
spinal cord and ganglia of the zebrafish
By JUDITH SHULMAN WEIS 1
From the Department of Biology, New York University
INTRODUCTION
In teleost fishes, unlike many other vertebrates, the spinal cord originates as a
solid structure, the neural keel, which subsequently hollows out. Unlike vertebrates in which the neural tube is formed from neural folds, and where the
neural crest arises from wedge-shaped masses of tissue connecting the neural
tube to the general ectoderm, teleosts do not possess a clear morphological
neural crest. Initially, the dorsal surface of the keel is broadly attached to the
ectoderm as described by Shepard (1961). As the neural primordia become larger
and more discrete, the region of attachment narrows, and cells become loose
(the 'loose crest stage'). These cells represent the neural crest. Subsequently they
begin to migrate and to differentiate into the various derivatives of neural crest.
Both sensory and sympathetic neurons arise from neural crest. At the time of
their migration the cells are not morphologically distinguishable. Only after
the sensory cells have aggregated into the primordia of the dorsal root ganglia
do they become morphologically identifiable as nerve cells, by the oval shape
of the cell body, appearance of neurofibrils, and outgrowth of axon fibers
(Levi-Montalcini, 1964). The subsequent growth of the ganglia is due to addition
of more migrating cells, mitotic activity of cells within the ganglia, and size
increase of individual cells as they mature. Balfour (1877) described the cephalocaudal development of spinal ganglia in Elasmobranchs from two club-shaped
masses of cells at the summit of the spinal cord or crest, which migrated ventrally
maintaining contact with the cord. Harrison (1901) observed a similar development of spinal ganglia in Salmo salar, although he believed that the cells migrated
out from the spinal cord.
Detweiler (1937) vitally stained the neural crest of Amblystoma with Nile blue
sulfate, and observed the migration of cells and condensation to form ganglia.
Weston (1963) observed this migration in the chick by radioactively labelled
1
Author's address: Department of Zoology and Physiology, Rutgers University, Newark,
New Jersey, U.S.A.
8
JEEM 19
110
J. S. WEIS
grafts of neural crest which developed into spinal ganglia, sympathetic ganglia,
melanoblasts, and Schwann sheath cells.
The precursors of the sympathetic nerve cells migrate from the crest somewhat
earlier than the sensory cells (Yntema & Hammond, 1947). At the time of their
migration they do not differ from other neural crest derivatives. When they have
aggregated in two columns dorsolateral to the aorta, they begin to resemble
nerve cells with elongated cell bodies and axons which show an affinity for
silver. Some cells move anteriorly and assemble in the head as the cervical
sympathetic ganglia. The sympathetic cells then undergo an increase in number
and size by mitotic activity and growth of individual nerve cells.
There has been considerable controversy in the past over the origin of the
sympathetic nervous system, with various investigators advocating spinal nerves,
spinal ganglia, neural tube, mesenchyme, and neural crest as possible sources.
The review by van Campenhout (1930 a) considers the various theories which
have been put forth. Conclusive evidence was obtained by van Campenhout
(19306), who experimentally investigated the development of the sympathetic
system in Rana. His results indicated that the sympathetic chain was derived
from the neural crest and had no contribution from the spinal cord. He found
that sympathetic elements could migrate longitudinally beyond the limits of the
segments from which their primary migration took place. Therefore, if a short
length of neural crest was removed, cells from adjacent normal levels moved
into the depleted area and grouped into ganglia. The animal therefore had no
spinal ganglia in the operated region but did have sympathetics. This is one of
the reasons for the confusion in the literature.
In teleosts the chain extends as far forward as the trigeminal nerve. The
preganglionic fibers for the head region arise from the spinal cord in the trunk
region and pass forward to the cranial sympathetic ganglia (Young, 1950).
The sympathetic systems of the teleosts Ambloplites rupestris, Micropterus
dolomieu and Perca flavescens were described by Huber (1900). In the trunk
portion, the chains are above the kidney and may be embedded within it. The
caudal portion is located within the hemal arch. Young (1930) described the
sympathetic system of Uranoscopus scaber, a teleost which is unusual in that no
pigment is associated with the sympathetic chains. In the anterior trunk region
the sympathetic ganglia lie close to the spinal ganglia with short rami communicantes. In the posterior trunk region the chains approach the midline, become
flattened between the dorsal aorta and cardinal vein, and become diffuse. Passing
backward between the kidneys, the two chains fuse and pass as a single cord
to the end of the abdomen. However, when they enter the hemal canal in the
caudal region they become paired again. In the caudal region the small ganglia
in each segment are connected to the spinal nerve, and across the midline by
transverse commissures.
In developing amphibia and fish there exists a primary sensory system of
Rohon-Beard cells which functions prior to the development of the spinal
Zebrqfish nervous system. I
111
ganglia. Beard (1889, 1892) initially described transient dorsal cells within the
spinal cords of Lepidosteus, Raja, and other species. These cells also are found
just outside the spinal cord under the meningeal covering, as a result of migration,
and send processes to the skin. They are later cut off from the central nervous
system, but persist for some time lying outside the cord. Similar cells were
described by Fritsch (1886) in Lophius, and by Harrison (1901) in Salmo, who
described the cells as arising from the dorsolateral surface of the cord near the
limiting membrane.
Giant supramedullary neurons lying directly dorsal to the cord or in its median
dorsal fissure have been described by Dahlgren (1897), Sargent (1899) and Burr
(1928) in a wide variety of fishes, sometimes only in larval stages, and sometimes
in mature animals as well.
Tracy (1961) found that the Rohon-Beard cells degenerated early in the
toadfish, Opsanus tau, the spinal ganglia developing at the time of hatching.
In the early swimming stages of the amphibian Amblystoma these cells are the
only sensory system (Coghill, 1914). Hughes (1957) described migration of
Rohon-Beard cells in Xenopus from within the cord to an extramedulary position. These cells serve as the afferent trunk system for 200 h of free swimming
life, after which time they are replaced by the dorsal root ganglia.
This paper describes the normal morphology and development of the spinal
cord and ganglia of the zebrafish, Brachydanio rerio (Hamilton-Buchanan), as a
study preliminary to experimental modification of their development. The zebrafish, also known as the zebra danio, is a member of the family Cyprinidae and is
native to India. This species is oviparous and can be bred easily, but capriciously,
in captivity. The egg is transparent and about 0-6 mm in diameter. Development is rapid—only 96 h from fertilization to hatching—at 26 °C. The normal
development of B. rerio has been described by Hisaoka & Battle (1958) and
Hisaoka & Firlit (1960), who divided the embryological period into a series of
stages. This species has been used extensively in experimental embryology.
MATERIALS AND METHODS
Stocks of mature zebrafish are easily maintained in the laboratory in spring
water, and eggs can be obtained throughout the year. Fish are spawned in tanks
with a net of plastic screening near the bottom. The eggs, which are demersal,
drop through the holes in the screening and thus cannot be eaten by the cannibalistic parents. The eggs are subsequently removed from the tank with a syringe.
In nature, the normal time of spawning is conditioned by light and occurs
normally at dawn. Using the method of Legault (1958), spawning tanks were
covered at night and uncovered in the morning. Spawning generally occurred
shortly after the tanks were uncovered. Eggs can be obtained from an individual
female once every 5 or 6 days (Hisaoka & Firlit, 1962). One mature female can
lay a few hundred eggs at a time. There is, however, a significant mortality
during development, the most critical period being that of gastrulation.
8-2
112
J. S. WEIS
To see the normal development of the nervous system in this species, eggs at
various stages of development were fixed in Bouin's fluid. The outer chorion
was removed with fine forceps before dehydration in a graded series of alcohols
to xylene. They were then embedded in paraffin, serially sectioned at 10/* and
stained with Delafield's hematoxylin and lightly counterstained with eosin.
Young fry were treated likewise. Some fish were raised for 6 weeks, by which
time they had reached different lengths. They were then fixed, sectioned and
stained as above. From each fish, four spinal ganglia were measured: three from
the anterior end of the spinal cord and one from the posterior end of the coelomic cavity. To determine the volume of a three-dimensional structure, uneven in
shape, reconstructions were made. The sections were projected to give a magnification of x 460, and the outline of the ganglion traced on paper for each
section in which the ganglion appeared. The outlines of all sections of the
ganglion were cut out of the paper and weighed on a balance. Weights of left
and right ganglia were pooled for each ganghon weight. This weight of the paper
is, or course, relative, but it reflects the mass of the ganglia. This method is
subject to a certain amount of error—variation in thickness of the paper, variation in weight of paper of the same area due to different humidity, inaccuracy
in cutting, etc. To investigate the extent of these sources of error, sixteen pieces
of paper of the same size were weighed and compared with each other. Their
weights on a very humid day were compared with those after being placed in a
drying oven for 45 min. The original variation in weight was 0-01 g in 0-60 g,
or about 1-5 %. After drying the greatest change was 0-2 g, or about 3 %. To
estimate my errors in tracing and cutting, eighteen ganglia were retraced, recut
and reweighed, and the average error was 4 %.
Cells were counted in one ganghon per fish (ganglion 2). The counts were
generally done under x 750 magnification, and each nerve cell whose nucleus
was present was counted on each section. Left and right counts were pooled.
At a different date, the cells were recounted without reference to the earlier
number. The largest discrepancies were about 10 %, and the average error was
considerably smaller. To correct for the spread of nuclei between sections, the
Floderus correction formula as described in Ebbeson & Tang (1965) was applied.
In this same ganglion the nuclear size was measured with a calibrated ocular
micrometer. In sections through the middle of the ganglion, the greatest diameter
of the nuclei of the majority of the larger neurons was measured.
RESULTS
In this study of the development of certain parts of the nervous system,
samples were chosen to correspond to the stages of Hisaoka & Battle (1958).
At stage 18 (14 h at 26 °C.) the solid neural keel is forming above the notochord,
which is flanked by a mesodermal sheet on either side (Plate 1, fig. A). The
cavity first appears in the brain at stage 20 (24 h), when the optic vesicles are
/. Embryol. exp. Morph., Vol. 19, Part 2
PLATE 1
All sections cut at 10/t, stained with hematoxylin and eosin.
Fig. A. Stage 18 (14 h). Axis formation; the neural keel is forming dorsal to the notochord.
K, Neural keel; N, notochord; Y, yolk.
Fig. B. Stage 21 (27 h). The neural tube is well-defined and hollow, with broad attachment
to the dorsal ectoderm. C, Neural crest; N, notocord.
Fig. C. Stage 24 (72 h). White matter is free of cells, migration of neural crest has occurred;
melanin pigment visible. P, Melanin pigment.
Fig. D. One week after hatching; Rohon-Beard cells are seen lateral to the spinal cord. RB,
Rohon-Beard cells.
j . s. WEIS
facing p. 112
J. Embryol. exp. Morph., Vol. 19, Part 2
Fig. E. Two and a half weeks after hatching. Rohon-Beard cells still visible laterally, but
spinal ganglia forming dorsally. G, Spinal ganglia; RB, Rohon-Beard cells; M, Mauthner's
fibers.
Fig. F. Mature animal. Section through trunk region showing typical position of spinal
ganglia. G, Spinal ganglia.
Fig. G. Mature animal. Section through cervical region showing the ventral position of the
ganglia in this portion of the body, and the thick neural arch cartilage dorsal and lateral to
the spinal cord. A, Neural arch; G, spinal ganglia; R, roots of spinal nerve.
Fig. H. Mature animal. Section through caudal region showing sympathetic ganglia ventral
to the aorta; commissure connecting ganglia is visible. S, Sympathetic ganglia.
j . s. WEIS
Zebrafish nervous system. I
113
forming. At stage 21 (27 h) the cord itself has become hollow, and the notochord
is becoming vacuolated (Plate 1,fig.B). The cells concentrated around the small
neural canal are the forerunners of the ependymal layer. At this stage the optic
cup has formed and lens induction has taken place. At stage 22 (37 h) a distinction can be seen between the white and gray matter, the cells becoming concentrated in the interior of the cord. The neural canal is quite ventral in position.
By this time otic vesicles have formed. At stage 23 (50 h) some neural crest cells
have migrated out, and melanin pigment is seen around the spinal cord. Initially
the melanin is densest immediately dorsal to the spinal cord at the original site
of the crest cells. The cells then migrate laterally and ventrally. At stage 24
(72 h) the gray matter of the cord is filled with large undifferentiated cells
(Plate 1, fig. C). This appearance is retained through the time of hatching.
The Rohon-Beard cells are first distinguishable at stage 22-23. They are initially
in lateral positions within the cord, but are subsequently found just outside the
cord under the meningeal covering as a result of migration. By stage 24 cells
were seen chiefly in this extramedullary position. They persist for 3-5 weeks as
small groups of cells lateral to the cord, between the two meningeal coverings
(Plate 1,fig.D). No supramedullary cells, as described by Sargent (1899), were
ever seen in this species. The cells are subsequently obscured by the connective
tissue cells which surround the cord to form the neural arches.
By the time of hatching, Mauthner's fibers are seen ventrolateral to the central
canal of the spinal cord. These large axons run posteriorly in the spinal cord and
are traceable to the caudal end of the spinal cord.
By 2 weeks after hatching, the spinal cord is approaching the mature condition.
The gray matter becomes organized in the shape of an inverted Y, the two dorsal
horns lying so close together that there is hardly any white matter between them
(Plate 2, fig. E). The small, oval-shaped neural canal is ventral in position and
surrounded by an ependymal layer. The large Mauthner's fibers lie ventrolateral
to it. The large ventral horn cells with large granular nuclei and conspicuous
nucleoli have a net of processes extending outward. The largest cells are most
frequently found medially, near the neural canal. They have a large amount of
cytoplasm, and nuclei about twice the size of those of neighboring cells. Ventrolateral motor neurons were generally somewhat smaller than mediodorsal ones.
At 2-4 weeks after hatching the spinal ganglia first appear as localized
condensations of neural crest material dorsolateral to the cord. The cells are at
first undifferentiated, appear mesenchymal, and are not clearly separable from
connective tissue. Subsequently, the cells take on a bipolar appearance, and the
ganglia become well-defined and clearly separated from surrounding tissue. It
is not possible to give an exact timetable for the development of spinal ganglia,
since their rate of development is determined by the growth rate of the individual
animal, which can vary extensively among animals of the same brood. Therefore, the state of development of the nervous systems of two animals of the same
brood which have reached different sizes will be correspondingly different. If an
114
J. S. WEIS
animal is stunted, not only is the rate of body growth slowed down, but also the
rate of development and differentiation of organ systems. In litter-mates all
sacrificed at the same date, it may be possible to see a wide spectrum of developmental stages, from those with remaining Rohon-Beard cells to those with
well-differentiated ganglia.
When first forming, the ganglia often appear as thin rows of cells along the
lateral and dorsolateral surfaces of the cord. They are sometimes thicker at
their ventral ends, at the site of the Rohon-Beard cells, indicating that perhaps
the Rohon-Beard cells may be incorporated into the spinal ganglia. It would
appear as if the neural crest cells migrate ventrally until they reach the RohonBeard cells, and then add on to them dorsally so that there is temporarily a single
composite structure. In other cases, however, there is a clear separation between
the Rohon-Beard cells laterally and the developing spinal ganglion dorsally;
occasionally there is a thin connexion between them (Plate 2, E). In older
specimens, however, the 'bulge' at the ventral end of the ganglion is never
present, all ganglia tapering sharply at their ventral ends. This would seem to
indicate that the original Rohon-Beard cells have disappeared, and that the cells
in the mature ganglion are only those derived from the neural crest several
weeks after hatching.
The ganglion cells themselves are large and elongate, with large nuclei,
distinct basophilic nucleoli (usually one, but sometimes two) and tapering cytoplasm with basophilic Nissl substance typical of nerve cells. Many cells retain a
bipolar appearance when mature. This has been previously noted in fishes by
von Lenhossek (1892) and Martin (1895), and is in contrast to other vertebrates
in which all spinal ganglion cells become unipolar when they mature. The
nuclei are spherical or oval in shape, and elongated along the plane of elongation
of the entire ganglion (roughly dorsoventral). The ganglia themselves are
elongate, roughly tear-drop-shaped, and are typically found dorsolateral to the
cord, wedged in between it and the muscle mass (Plate 2, fig. F). The ganglia
often extend upward, far beyond the dorsal end of the spinal cord. However,
the first three spinal ganglia are large, round, and lie ventrolateral to the spinal
cord, like more typical vertebrate spinal ganglia. These three ganglia are found
in the cervical region, where the vertebrae are provided with unusually thick
neural arches (Plate 2, fig. G). The ganglia are located below the ventral ends of
the neural arches. The ganglia chosen for examination were the first three pairs
posterior to these ventrally located ones; that is, the first three pairs of 'typical'
spinal ganglia. The first of these, which is designated as 'ganglion number 1',
appears just posterior to the termination of the thick neural-arch cartilage which
had been occupying the space dorsal and lateral to the spinal cord. This first
ganglion is frequently somewhat intermediate in position, usually long and thin,
and not extending as far dorsally as the succeeding ganglia, which were more
tear-drop-shaped. The other ganglia chosen for examination were located at the
level of the cloaca. In an occasional fish the ganglia at the level of the cloaca and
Zebrafish nervous system. I
115
some located farther caudally were situated in the ventral position. These
ganglia however, were still elongated and tear-drop-shaped, but their orientation
was 180 ° from that of the typical ganglia. Therefore, they do not resemble the
much larger, round, ventrally located ganglia in the cervical region. In one
specimen, one member of the pair was dorsally located while the other was
ventral. In a few specimens, cells were concentrated at both ends of the ganglion,
Table 1. Weights of paper reconstructions, corrected cell counts, and
nuclear diameters of ganglia from Brachydanio rerio of different lengths.
Standard
length*
(mm)
4
6
8
10
14
16
Nuclear
diameter %
Ganglion
1
2
3
Cloacal
1
2
3
Cloacal
1
2
3
Cloacal
1
2
3
Cloacal
1
2
3
Cloacal
1
2
3
Cloacal
Weight
S.E.
7-6
7-3
1-7
1-5
9-4
7-2
80
7-5
151
170
16-6
18-4
20-4
25-3
27-1
28-8
59-5
59-5
56-5
570
11
0-9
0-6
0-9
0-9
1-5
1-2
1-8
1-5
2-5
1-9
2-8
3-5
4-5
0-5
30
50
8-5
30
90
850
68-5
980
980
Cell count f
S.E.
141
0-8
2-4-3-6
24-9
2-8
3-6^-8
45-2
5-7
4-8-6-0
61-8
2-6
6-0-7-2
900
150
7-2-8-4
90-8
110
7-2-8-4
* Standard length—snout to caudal peduncle.
t Based on all neurons whose nuclei were present, counted in all sections in which the
ganglion appeared. Counts for left and right pooled, then corrected.
% Based on maximum-sized nuclei contained in sections through the middle of the ganglion,
in all fish.
and were sparse in the middle, indicating great variability of these caudally
located ganglia. In the vast majority offish examined, however, the cloacal and
caudal ganglia retained the typical dorsal position.
In contrast with the development of the spinal ganglia, the sympathetic
system makes its appearance somewhat later in development, a month or so
116
J. S. WEIS
after hatching. In the trunk portion of the body, the two sympathetic chains
lie lateral to the aorta and are embedded in the kidney, and therefore extremely
difficult to see. In the caudal portion of the body the sympathetic chains lie
within the hemal canal, ventral and lateral to the aorta. The small ganglia on
each side are connected to each other by transverse commissures (Plate 2,
Fig. H). The ganglia are quite small, and favorable sections could not be obtained
in many specimens. Frequently melanin obscured the ganglia. Consequently,
most attention was devoted to the spinal ganglia.
The data from paper reconstructions of spinal ganglia from fish of different
sizes are presented in Table 1. In 4 mm fish, ganglia averaged 7-5; in 6 mm fish
they averaged 8-0; in 8 mm fish, 16-8; in 10 mm fish, 25-4; in 14 mm fish, 58-1;
and in 16 mm fish, 85-1. These data indicate that as the fish grow, the ganglia
grow in proportion to the size of the fish. Fish size would therefore appear to be
more important than age in determining ganglion size.
The cell counts and measurements of nuclear diameters of cells in ganglion 2
are also presented in Table 1. Ganglia from 4 mm fish averaged 14-1 cells,
(corrected value) 6 mm fish averaged 24-9 cells, 8 mm fish averaged 45-2 cells,
10 mm fish averaged 61-8 cells, 14 mm fish averaged 90-0 cells, and 16 mm fish
averaged 90-8 cells. Through this size range, nuclear diameters increased from
2-4 to 8-4 fi. These data indicate that the number of cells in the ganglia increases
with the length of the fish, and that the size of the ganglion cells likewise
increases with fish size.
DISCUSSION
It has been observed that in the developing zebrafish the Rohon-Beard cells
persist for several weeks after hatching, and that the spinal ganglia form relatively late. This is probably related to the fact that the fish hatch in only 4 days
and are still in a very immature state at that time. In contrast, Fundulus takes
approximately 2 weeks to develop, and hatches in a much more advanced state,
with its spinal ganglia already formed (personal observation). It might be
considered that many of the same developmental events are taking place in
Fundulus before hatching as in Brachydanio while already actively swimming
around.
The typical dorsolateral position of the spinal ganglia in the zebrafish is
different from that in other classes of vertebrates, and from that in a number of
other teleosts which I have examined, namely Fundulus, Rivulus and Pterophyllum, in which the ganglia occupy a more ventral position relative to the cord. In
Fundulus the ganglia are well formed by the time of hatching. Photographs
accompanying the papers of Ray (1950) and Tracy (1961) indicate that in
Lampanyctus and Opsanus the ganglia likewise occupy ventral positions. However, Rhinichthys (the dace, also in the family Cyprinidae) has ganglia positioned
as the zebrafish does, as do the closely related members of the same family, the
Zebrafish nervous system. I
111
pearl danio {Brachydanio albolineatus) and spotted danio (B. nigrofasciatus)
(personal observation).
From the data on ganglion size, cell number and nuclear diameters, several
facts can be discerned. One can observe that as the fish grow the ganglia grow,
and there is continual increase in cell number, in addition to size increase of
cells during this growth period. This phenomenon might be considered to be
related to the prolonged immature condition of the nervous system in this
species. This continual increase in cell number in the fish ganglia is in contrast
to the situation in some other vertebrates such as birds and mammals in which
the number of spinal ganglion cells reached in embryonic stages remains constant through the rest of the animal's life, and growth of spinal ganglia is
achieved mainly through hypertrophy of cells already present. Mitoses were not
observed in the zebrafish spinal ganglia, so it would seem likely that there is a
continual addition and differentiation of new cells, rather than multiplication of
already differentiated nerve cells.
SUMMARY
1. In embryos and young fry of the zebrafish, Brachydanio rerio, the sensory
function of the nervous system is fulfilled by a primitive transitory system of
Rohon-Beard cells. These are replaced 2 or 3 weeks after hatching by the
developing spinal ganglia.
2. The spinal ganglia are typically tear-drop-shaped and occupy a dorsolateral position relative to the spinal cord, with the exception of the first three
pairs of ganglia which are large, round, and lie ventrolateral to the cord.
3. As the fish grow, the spinal ganglia grow in size, due to an increase in cell
size, and an increase in cell number. This is different from the situation in
mammals and chicks, for example, in which the number of cells reached in
embryonic stages remains constant, and growth of ganglia is achieved only by
means of cell enlargement.
RESUME
Analyse du developpement du systeme nerveux du Poisson-zebre, Brachydanio
rerio. I. La morphologie et le developpement normaux de la moelle epiniere
et des ganglions rachidiens
1. Chez les embryons et les jeunes alevins de Brachydanio rerio, les fonctions
sensorielles du systeme nerveux sont assurees par un systeme primitif transitoire
de cellules de Rohon-Beard. Celles-ci sont remplacees par les ganglions rachidiens en cours de developpement, deux ou trois semaines apres l'eclosion.
2. Les ganglions rachidiens ont une forme larmee typique et se trouvent en
position dorso-laterale par rapport a la moelle epiniere, a l'exception des trois
premieres paires de ganglions, qui sont grands, arrondis et situes ventrolateralement a la moelle.
3. Au fur et a mesure que le poisson grandit, la taille des ganglions rachidiens
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J. S. WEIS
s'accroit, en raison d'une augmentation de la taille et du nombre de leurs cellules.
C'est la une difference avec la situation realisee par exemple chez les mammiferes
et les oiseaux, oil le nombre de cellules atteint au cours des stades embryonnaires
reste constant, et ou la croissance des ganglions est realisee seulement par
l'accroissement de la taille des cellules.
I wish to express my appreciation to Dr Alfred Perlmutter for his interest and helpful
suggestions throughout the course of this study. I wish to thank Dr Elmer Bueker for his
advice and use of his equipment. My appreciation is extended to my husband, Dr Peddrick
Weis, for the use of laboratory space and for his constant interest and encouragement
throughout the course of this study. This work was supported in part by U.S.P.H.S. Grants
NB-05755 and NB-03979. This paper is part of a dissertation submitted in partial fulfilment
of the requirements for the Ph.D. degree at New York University.
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{Manuscript received 16 June 1967, revised 17 October 1967)