Download Formation of Axon to Myocyte Contacts in Drosophila Cell Cultures

AMER. ZOOL., 13:331-336 (1973).
Formation of Axon to Myocyte Contacts
in Drosophila Cell Cultures
ROBERT L. SEECOF, J. JAMES DONADY, AND PILAR TORIBIO-FIORIO
Department of Developmental Biology, City of Hope National Medical Center,
Duarte, California 91010
SYNOPSIS. Cultures of Drosophila embryonic cells offer new opportunities for studying
myoneural junctions. In culture, neuroblasts and myoblasts differentiate and yield
neurons and myocytes. Some axons grow across the surface of the culture vessel and
attach to myocytes, forming functional myoneural junctions. Therefore, all stages in
junction formation may be examined in vitro under conditions where pharmacological,
electrophysiological, and other commonplace approaches are facilitated. This method
offers an additional, most powerful approach for studying the junctions, that of genetic analysis. Drosophila mutations may be sought which affect junction formation
and function. Altered cells and junctions from mutants may then be compared to
those from wild-type animals in order to dissect the gene-directed steps underlying
junction phenomena.
INTRODUCTION
The term myoneural junction refers to
a place of contact between nerve and
muscle where the motor impulse is transmitted to the muscle. The microscopic site
of such a junction is formed by an axon
terminus which is closely applied to the
membrane of a muscle cell or a muscle
syncytium, and all of the cellular specializations associated with the site are
usually considered to be part of the junction. Thus, the myoneural junction includes the presynaptic membrane of the
axon terminus with its associated axoplasm
containing synaptic vesicles and mitochondria, the area of the sarcoplasmic (postsynaptic) membrane opposite the presynaptic membrane, and the narrow synaptic
cleft between the two membranes. Myoneural junctions with this general morphology are characteristic of both vertebrates
(Coers, 1967) and insects (Smith and Treherne, 1963), which suggests an ancient
common evolutionary origin and a common modus operandi for these junctions in
present-day animals.
This work was supported by N.T.H. grants
NS09330 and AI05038 to R. I.. S. and by a fund
established in the name of the Poultry Industry
Research Fellowship honoring Richard Popik.
Present address of J. James Donady: Department of Biology, Wesleyan University, Middletown,
Connecticut 06457.
Through the efforts of many investigators, it has been established that the axonic signal travels to the axon terminus,
where it causes the release of chemical
transmitter into the synaptic gap. The
transmitter then interacts with the postsynaptic membrane to excite muscle concontraction or, in the case of inhibitory
transmitters of some invertebrates, to retard muscle contractions.
There is an imposing literature describing the electrical phenomena and the
fluxes of inorganic ions that accompany
axonal and myoneural transmission and
the response of the sarcoplasmic membrane. Studies of the pharmacology and
detailed morphology of myoneural junctions have been numerous too, and today
we have a wealth of information concerning all these aspects of junctional transmission. However, many basic questions
are still only partially answered and others have not yet been approached.
For example, what characteristics of presynaptic and postsynaptic membranes give
them the abilities to transport or bind
transmitter? At what stages of cell differentiation do neurons and muscle become
competent to form myoneural junctions?
What guides embryonic cells to make proper connections, and thus later initiate adaptive movements? What determines the
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SEECOF, DONADY, AND TORIBIO-FIORIO
functional characteristics of each junction
and what is the sequence of steps by which
the junction differentiates?
These questions were selected from the
many the could be posed, because answers to them may soon be forthcoming.
These questions and others like them can
now be approached through the use of
Drosophila cell cultures. The remainder of
this article will describe the formation of
myoneural junctions in such cultures and
indicate how the in vitro system may be
used to study the junctions further. The
ullrastructural characteristics of a myoneural junction from Drosophila cell culture
have already been reported (Seecof et al.,
1972). The present report will deal with
light-microscope observations of axons
growing across the bottom of the culture
dish and contacting myocytes, and with relationships among the differentiating neurons and myocytes during this happening.
EXPERIMENTAL
Drosophila melanogaster was used for all
experiments. Cells can be taken from
Drosophila gastrulae about 30 min after
the initiation of gastrulation, before overt
cell differentiation has begun and cells differ visibly only by size. A single embryo of
this stage yields a culture of about 4000
cells attached to the substrate as single
cells or small aggregates. Among the cells
are neuroblasts and myoblasts, and during
the next several hours these differentiate
into neurons and myocytes, respectively.
Cells from a single embryo yield about 200
axons longer than 50^, and a few hundred
mononucleate myocytes. Many myocytes
form aggregates and some of the aggregates
may prove to be myotubes. Technique for
making cultures and evidence for the above
statements can be obtained from Seecof et
al. (1971, 1972) and Seecof and Donady
(1972).
Axon contacts to myocytes can be seen
by light microscope (Seecof and Unanue,
1968; Seecof et al., 1972). If an axon attached to a myocyte is stimulated electrophysiologkallv, the imotvte is usually
caused to contract. In a series of trials, 85
per cent of axons tested caused the attached myocytes to contract and the true
percentage of functional junctions may be
even higher (Seecof et al., 1972). These
trials demonstrated the presence of functional neurons, myocytes, and myoneural
junctions in the cultures.
Individual neuroblasts have been observed during their differentiation in vitro.
One kind of neuroblast yields approximately 16 daughter cells, termed ganglion
cells, and at least one, and probably all, of
these daughters are neurons. Divisions occur rapidly and neurons with cell bodies
of 2-5//. in diameter and axons greater than
50ju long are elaborated in vitro between
7 and 16 hr after the initiation of gastrulation. All cell ages quoted in this article
are calculated from the onset of gastrulation in the donor embryo, rather than the
time of culture initiation. Details of this
neuroblast differentiation will be published
elsewhere (Seecof and Donady, unpublished). Other kinds of neuroblast differentiation in the cultures are not excluded.
Individual myoblasts have been observed
during their differentiation in vitro. One
kind of myoblast divides once at about 5
hr. The daughter cells, which we call myocytes, elongate and aggregate beginning at
about 12 hr, arrange themselves in a roughly linear array, and then often show spontaneous contractions. A note concerning
myoblast differentiation has already been
published (Seecof and Donady, 1971) and a
full report is in preparation. Other kinds
of myoblast differentiation in the culture
are not excluded.
Evidence published thus far proves that
neurons, myocytes, and junctions differentiate in vitro, but the manner in which
axons contact myocytes has not been reported. It seemed possible that stem cells
which were destined to be joined by myoneural junctions in vitro were in contact
when they settled to the dish surface. Cell
differentiation would be required for junctions to form, but instead of axon growth
to the imotUc, axons would merely elongate IxHweeu the attached cells.
AXON TO MYOCYTE CONTACTS
FIG. 1. Steps in myoneuronal junction formation.
a, Age of cells, 5 hr 35 min. (Age of cells calculated from the time of gastrulation initiation in
the donor embryo. Cells taken from the donor embryo 30 min after the initiation of gastrulation.)
Neuroblast in division (N), Myoblast (M), Myo-
333
cyte (m) . b, Age of cells 7 hr 50 min. c, Age of
cells 10 hr 40 min. d, Age of cells 12 hr 20 min.
e, Age of cells 13 hr 5 min. /, Age of cells 13 hr
45 min. g, Age of cells 24 hr 20 min. h, Age of
cells 34 hr 25 min. (Phase contrast. Magnification: 435 X-)
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SEECOF, DONADY. AND TORJBIO-FIORIO
The latter possibility is still conceivable,
but observations of differentiating cells
show it to be very unlikely. Several instances have been observed of axons growing to myocytes and making attachments,
and there is no reason to doubt that all the
axons attaching to myocytes grow to their
destinations. A representative sequence of
differentiating cells is given in Figure \a
through ]h.
The cells shown in Figure 1 were taken
from an embryo 30 min after the onset of
gastrulation and dispersed in a plastic culture dish. They were observed and photographed with a Zeiss inverted microscope
using a 40X objective and phase contrast
illumination. This sequence of photographs shows differentiating neurons and
myocytes, but does not illustrate all the
blast cell divisions and associated phenomena referred to above. The sequence
was chosen to illustrate axon growth to
myocytes, and other events can be seen only
fragmentarily.
Figure la shows cells at 5 hr 35 min.
Three myoblasts are at the upper right,
and below them are two myocytes derived
from a myoblast division. The three cells
grouped at the center are daughter cells
from neuroblast divisions. At the upper
left, one or more neuroblasts are in division. These cells are rounded and above
the plane of focus. Cellular detail cannot
be seen unless a cell is flattened in the
plane of focus.
Figure 1 b shows cells at 7 hr 50 min.
Myoblasts have completed divisions and,
thus, become myocytes. Neuroblast divisions have continued and it is not possible
to count the number of daughter ganglion
cells. Cell processes probably representing
axons are appearing from the ganglion
cells. Myocyte processes are somewhat longer.
Figure \c shows cells at 10 hr 40 min.
Axons are present and axon terminals contact both groups of differentiating myocytes. Some myocytes have extended filaments up to 15 /am long. Perhaps diese
serve to contact other myocytes and provide a means to guide the myocytes in their
ensuing aggregation.
Figure Id shows cells at 12 hr 20 min.
Myocytes are beginning to elongate, aggregate, and align. Axon contact with myocytes has probably been lost.
Figure \e shows cells at 13 hr 5 min.
Myocyte elongation, aggregation, and
alignment is progressing. Axon contact to
myocytes is present.
Figure 1/ shows cells at 13 hr 45 min.
Myocyte elongation, aggregation, and
alignment is progressing. The roughly
linear array is suggestive of the strings of
cells observed during the aggregation of
chick cells to form myotubes in vitro (Konigsberg, 1963). Axon contact to myocytes
has probably been lost.
Figure \g shows cells at 24 hr 20 min.
Myocyte elongation, aggregation, and
alignment is completed. The myocytes have
apparently fused to form a myotube. Axon
attachment is now stable and the unattached "branches" are withdrawing. As
mentioned above, junctions of this age and
appearance are nearly always functional.
Figure \h shows cells at 34 hr 25 min.
Myocytes have partially separated. Either
they had never fused or the myotube is
separating into myocytes. The latter event
has been reported for chick myotubes in
vitro (Cooper and Konigsberg, 1959). The
separation was unusual and probably was
caused by a deterioration of culture conditions. The attachment to muscle is now apparently by way of a bundle of axons, a
nerve.
DISCUSSION
The phenomena seen in Figure 1 are
typical for cells in cultures like these.
Axon growth is dynamic, with branches extending ancl withdrawing, as is well known
for vertebrate axons in vitro. Contact often
is made between an axon from a group of
ganglion cells and a myocyte, or between
axons from two groups of ganglion cells.
When such contact is made, the axonic
connection often becomes large in diameter. As in Figure \h, the connection usually appears multistranded and, therefore,
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AXON TO MYOCYTE CONTACTS
probably was formed by additional axons
growing alongside the pioneer axon. Electron microscopy will be required to confirm that such connections are not huge
single axons. Even if such connections are
indeed proper nerves, we couldn't immediately conclude that axon growth was guided by the successful synaptic contact. It
may be that other causes constrained the
axons to grow in that direction.
Myocytes are visibly motile, and neurons
possibly have a limited motility. When two
incompletely differentiated cells meet and
then separate, they often retain contact by
a very fine tendril. It is possible that the
tendril is sometimes too fine to discern
with a 40X objective. The latter consideration is important to the discussion of
axon-myocyte contact, such as is shown in
Figure 1.
Axon contact is first shown in Figure
\c, before the myocytes are elongated or
pulsatile. Pulsations have never been detected before 13 hr. The decision as to
whether contact was present was made by
direct observation, which is more reliable
than photographic reproduction. Afterwards, the contact were apparently lost, regained, lost, and finally regained. This suggests a dynamic interaction between axon
and myocyte, but we cannot be sure that
losses of contact were real. An important
question here is: Are there discrete developmental stages where neurons and myocytes become capable of establishing junctions? This question will be answered by
challenging axons with differentiating
myocytes and at the same time varying neuron and myocyte ages.
A related question is: Is the direction of
axon growth random or is it somehow oriented toward myocytes? The question
probably can be answered by scoring the
directions of axon growth in these cultures,
but such scoring has not yet been done.
We noted in the Introduction that
Drosophila cell cultures could be employed
to approach certain questions concerning
the myoneural junction. Indeed, we have
already considered two of them: the question of cell differentiation stage versus com-
petency to form junctions, and the question as to whether axon growth is directional. Problems concerning the sequential
steps of junction formation can readily be
approached by use of this material. The
junctions are not shielded by unrelated
cells, and this fact favors pharmacological,
electrophysiological, and electron microscopical studies.
The cultured cells can be used in these
various ways, and they can also be used in
another, most powerful way to answer fundamental questions concerning myoneural
junctions. That is, junction phenomena
can be studied through an analysis of mutants. It is possible to select mutations of
Drosophila that cause embryonic lethality
or altered behavior and maintain these mutations in recessive condition. If mutant
embryos gastrulate, their cells can be cultured and studied. Some of these mutations
should affect the in vitro cell and junction
differentiations reported above. Each mutant effect will give insight into junction
development and function. For example,
a mutant might be obtained which, in
vitro, gave axons that approached myocytes but failed to establish contact. This
would indicate that such contacts are directed by gene function, and mutant cells
could be compared to wild-type cells in order to elucidate the underlying biochemistry. Each mutation would represent a genedirected step in junction development or
function, and all the steps could eventually be defined. We are presently hunting
for mutations like these and hope to use
them in future investigations of the myoneural junction.
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