/ . Embryol exp. Morph. Vol. 67, pp. 89-100, 1982
Printed in Great Britain © Company of Biologists Limited 1982
gO,
Morphology of programmed cell death in
the ventral nerve cord of Caenorhabditis
elegans larvae
By ALISON M. G. ROBERTSON 1 AND J. N. THOMSON 2
From the Department of Pathology, University of Edinburgh Medical School,
and MRC Laboratory of Molecular Biology, Cambridge
SUMMARY
In the nematode C. elegans, cells undergoing programmed death in the developing ventral
nerve cord were identified by Nomarski optics and prepared for ultrastructural study at
various times after their birth in mitosis.
The sequence of changes observed suggests that the hypodermis recognizes the dying cell
before completion of telophase. The dying cell is engulfed and digestion then occurs until all
that remains within the hypodermal cytoplasm is a collection of membranous whorls interspersed with condensed chromatin-like remnants. The process shares several features with
apoptosis, the mode of programmed cell death observed in vertebrates and insects. The
selection of cells for programmed death appears not to involve competition for peripheral
targets.
INTRODUCTION
The morphology of programmed cell death has been studied widely in the
morphogenesis of mammalian (Ballard&Holt, 1968; Farbman, 1968; Matthiessen
& Andersen, 1972; Schweichel & Merker, 1973; El-Shershaby & Hinchliffe,
1974, 1975), avian (Bellairs, 1961; Saunders, 1966; Saunders & Fallon, 1966;
Manasek, 1969; Hammar & Mottet, 1971; Schliiter, 1973; O'Connor &
Wyttenbach, 1974; Hurle & Hinchliffe, 1978), amphibian (Kerr, Harmon &
Searle, 1974), reptilian (Fallon & Cameron, 1977) and insect tissues (Goldsmith,
1966; Giorgi & Deri, 1976). Cell death in these systems appears to be by
apoptosis (Kerr, Wyllie & Currie, 1972; Wyllie, Kerr & Currie, 1980), a mode
of death which is characterized by condensation of nuclear chromatin, compaction of organelles, budding into membrane-bounded fragments and
phagocytosis by adjacent cells.
By contrast little is known about the ultrastructure of programmed cell death
in the morphogenesis of lower invertebrates, but the larvae of the nematode
1
Author's address: Department of Pathology, University of Edinburgh Medical School,
Teviot Place, Edinburgh EH8 9AG, U.K.
3
Author's address: MRC Laboratory of Molecular Biology, Hills Road, Cambridge,
CB2 2QH, U.K.
90
A. M. G. ROBERTSON AND J. N. THOMSON
C. elegans provide a uniquely favourable system in which to study this phenomenon. By the use of Nomarski differential interference contrast microscopy
on living C. elegans, all postembryonic cell lineages have been followed (Sulston
& Horvitz, 1977; Kimble & Hirsh, 1979). The precise time of division, migration and death is known for these cells with lespect to the stage of larval
development, and the position of each of these cells is also accurately defined.
In particular, at about the time of the first larval moult, 56 nerve cells are
added to the ventral nerve cord and associated ganglia by a uniform division of
13 neuroblasts (P0-P12) followed by a defined pattern of cell deaths (Sulston &
Horvitz, 1977). We here record a transmission electron microscope study of
progeny of P l l which are programmed to die (Fig. 1). The fate of these cells
is traced from birth at mitosis through to death and disappearance.
MATERIALS AND METHODS
Culture. Self-fertilizing hermaphrodite stocks of Caenorhabditis elegans var.
Bristol (Strain N2) were grown at 20 °C on agar plates seeded with Escherichia
coli OP50 as previously described (Brenner, 1974).
Microscopy. Our studies were restricted to cell death in the P l l lineage
series of nerve cells observed during the first and second larval stages after
hatching (LI and L2 respectively). These nerve cells are situated in the posterior
end of the nematode approximately 20 /tm from the anus. Cells whose death
was known from lineage studies to be either imminent or in progress were
identified and photographed by Nomarski optics (Sulston & Horvitz, 1977);
the animal was then immediately fixed for electron microscopy.
Preparation for transmission electron microscopy. The individual nematodes
were fixed in 1 % OsO4 in 0-1 M cacodylate buffer, pH 7-4, for 1 h at room
temperature. The nematode was bisected with a fine razor blade and the caudal
half treated en bloc in 1 % uranyl acetate in 0-05 M maleate, pH 6-1, for 45 min
(Ware, Clark, Crossland & Russell, 1975), and then transferred on to a thin
layer of 1 % agar and covered with a drop of 1 % agar which was then allowed
to set. A block of agar containing the specimen was then excised, dehydrated
and embedded in Araldite as previously described (Ward, Thomson, White &
Brenner, 1975). Sections of approximately 50 nm were cut with a diamond
knife on an LKB Ultratome III. Ribbons of serial section were cut transversely
to the long axis of the nematode, and were picked up on formvar coated 75
mesh grids. The sections were post-stained with 5 % uranyl acetate, for 10 min
at 60 °C, and lead citrate for 5 min and viewed and photographed in an AEI
EM 6B microscope.
RESULTS
Observations with Nomarski optics
P l l precursor nerve cells undergo divisions, migrations and deaths according
to the lineage already established (Sulston & Horvitz, 1977) (Fig. 1). The
Cell death in C. elegans
91
Time (h)
post hatch
0
Pll
Anterior
end of
nematode
Posterior
end of
nematode
LI
10
Moult
P l l .aap
1
20
-
PI 1 . aaap
L2
P l l . aaaa
P l l .apa
I
I
Nerve
cells
Moult
30
P l l . app
Pll . p
I Hypodermal
cell
1-
Fig. 1. PI 1 lineage in the post-embryonic development of the ventral nerve cord of
Caenorhabditis elegans. ' a ' and ' p ' represent anterior-posterior divisions of the
parent cell. When a cell divides each daughter is named by adding to the name of
its mother cell a letter representing its position immediately after division relative
to its sister cell. 'X' indicates cell death; 0 , nematode in lethargus.
surviving progeny are three nerve cells - P l l . aaaa, P l l . apa, P l l . app - and 1
ventral hypodermal c e l l - P l l . p. Cell nuclei and large nucleoli are resolved
clearly by Nomarski optics though cell boundaries cannot always be visualised
(Fig. 2). Nerve cells have granular nucleoplasm and generally no visible nucleolus whereas ventral hypodermal cells have large nuclei and nucleoli. Pll .aap
and PI 1. aaap undergo programmed death. In other regions of the ventral cord,
homologues of these cells survive and become motor neurons. The deaths of
P l l . a a p and Pll.aaap appear identical and our results and illustrations are
drawn from both.
Using Nomarski optics, morphological changes involved in programmed
death can be divided into five stages (I-V) (Fig. 2), which are described ultrastructurally in the following section. Division of the parent cell occurs (Stage I)
and soon afterwards the cytoplasm of the daughter cell programmed to die
seems to become less refractile (Stage II). The nucleus of the dying cell then
becomes less granular and more refractile and refractile beads appear around it
(Stage III). After some time the entire cell becomes highly refractile (Stage IV).
Sometimes Stage III—IV is episodic: the dying cell appears to be approaching
92
P
A. M. G. ROBERTSON AND J. N. THOMSON
-30min
Fig. 2. Birth and death of cell Pll .aap in the ventral nerve cord of one individual
LI larva; Nomarski differential interference contrast microscopy; left lateral
aspect; bar 10 jim. P, Parent PI 1. aa neuroblast (arrowhead); Stage I (late telophase)
to Stage V, sequential changes observed (timing indicated) during death of PI 1 .aap
(arrowed and indicated by the bracket). The other cell indicated by the bracket is
PI 1.aaa, the viable sister cell of Pll.aap. See text for details, nc, Nerve cell
nucleus; vh, ventral hypodermal nucleus; vnc, ventral nerve cord.
Stage IV but then reverts to Stage III or even Stage II for a while before eventually proceeding to Stage IV. After a few minutes the refractility of the perimeter diminishes; the central region remains refractile for longer but eventually
shrinks (Stage V) and disappears.
Figs. 3-13. Transmission electron micrographs of transverse sections through the
ventral nerve cord of the LI larva of Caenorhabditis elegans. Sections are cut
transversely to the long axis of the nematode. AV, Autophagic vacuole; BM,
basement membrane; C, cuticle; H, hypodermis; M, muscle cell; MIT, mitochondrion; NCN, nerve cell nucleus; NF, nerve fibres; NU, nucleolus; SP, spindle
tubules.
Cell death in C. elegans
93
Figs. 3-5. Sections through the PI 1 .aaa parent cell which is in telophase (Stage I).
The cell is dividing into Pll.aaaa (anterior daughter) (Fig. 3) and Pll.aaap
(posterior daughter) (Fig. 4). Note the early formation of the nuclear membrane in
both daughter cells. The presence of an intercellular bridge containing spindle
tubules is seen in a more central section through the dividing cell (Fig. 5). Both
daughter cells appear normal though Pll .aaap, the daughter cell programmed to
die, has an arm of hypodermis extending around it (Fig. 4, arrow), x 35000.
4
EMB
67
94
A. M. G. ROBERTSON AND J. N. THOMSON
/.%
Figs. 6 and 7. Sections through the daughter cells Pll .aaaa (Fig. 6) and Pll .aaap
(Fig. 7) formed by division of the Pll.aaa parent cell (StageII). The dying
Pi 1. aaap daughter cell is surrounded by the hypodermis which shows thin infolding
parallel processes (Fig. 7, arrows). The Pll.aaaa daughter cell has an arm of
cytoplasm extending into the region of the nerve fibres (Fig. 6, arrow), x 37600.
Cell death in C. elegans
95
Ultrastructural observations
The ultrastructure of the ventral nerve cord of the LI larva (Figs. 3-13) is
essentially the same as previously described for the adult C. elegans (White,
Southgate, Thomson & Brenner, 1976). It consists of a bundle of nerve fibres
which run longitudinally down the ventral mid-line of the nematode alongside a
ridge of hypodermis. The nervous and hypodermal tissues are enclosed within
a basement membrane which separates them from the muscle cells.
The cytoplasm of nerve cells is granular and contains mitochondria and
sparse endoplasmic reticulum. There are scanty aggregates of chrcmatin
distributed throughout the nucleoplasm and there is a small nucleolus. The
hypodermal cytoplasm is granular, rich in mitochondria and endoplasmic
reticulum, and shows a prominent Golgi complex; the nucleus is large and
contains a large nucleolus with scanty aggregates of chromatin throughout the
nucleoplasm.
Figures 3 and 4 show the birth of anterior/posterior daughter cells respectively from the parent cell, which is in telophase (Stage I); serial sectioning
reveals an intercellular bridge containing spindle tubules still connecting the
daughters (Fig. 5). The internal ultrastructure of the two daughter cells appears
normal, the only difference being that an arm of hypodermis is extending around
the daughter cell which is programmed to die (Fig. 4).
After division (Stage II), the two daughter cells (Figs. 6 and 7) still show no
difference in the structure of the nucleus and cytoplasmic organelles; however,
there are now thin parallel processes of hypodermal cytoplasm surrounding the
dying cell (Fig. 7). The anterior daughter cell (which in this case is a putative
motor neuron) can be seen extending an arm of cytoplasm into the region of the
nerve fibres - presumably the formation of a nerve axon; this is not seen in the
dying cell.
During Stage III (Fig. 8) the cytoplasm of the dying cell appears condensed
and the nuclear envelope is dilated. Nuclear chromatin forms granular aggregates, notably underlying the nuclear membrane, and in addition, a cluster of
electron-dense coarse particles appears in the centre of the nucleus, probably
representing an altered nucleolus. Sections through the posterior part of the
dying cell (Figs. 9-12) reveal that it splits into membrane-bounded fragments,
surrounded by what appears to be an arm of hypodermal cytoplasm separate'
from that which surrounds the major portion of the dying cell.
The dying cell in Stages IV-V (Fig. 13) shows an increase in whorling of
internal and plasma membranes, accompanied by irregularity of the cellular
outline. The mitochondria appear distorted and are frequently within autophagic vacuoles, and there is a reduction of cytoplasmic granularity. The
nuclear membrane becomes intensely convoluted and it is difficult to be certain
of its continuity in serial sections. However, some sections demonstrate prolific
budding of structures bounded by nuclear membrane and in addition, dense
4-2
96
A. M. G. ROBERTSON AND J. N. THOMSON
Figs. 8-12. Fig. 8. The dying PI 1 .aap daughter cell (Stage III) shows condensed
cytoplasm. The nucleus shows granular aggregates of chromatin and dilatation of
the nuclear envelope. A thin layer of hypodermis (arrow) surrounds the dying cell,
x 35300.
Cell death in C. elegans
97
Fig. 13. The dying Pll .aap daughter cell surrounded by the hypodermis (straight
arrow) shows membranous whorling with reduction of cytoplasmic granularity,
distortion of the mitochondria and the presence of an autophagic vacuole (Stage
IV-V). The nuclear membrane is breaking down and shows convolutions (curved
arrow); small fragments of condensed chromatin-like material are present, x 37500.
chromatin-like material is evident. Again, membrane-bounded fragments are
seen in the posterior part of the dying cell and these are enclosed within the
same arm of hypodermis as the rest of the dying cell.
As the dying cell shrinks (Stage V) there is complete breakdown of the
nuclear membrane and condensed chromatin-like fragments are still seen
within the cytoplasm. The latest recognisable appearance of the dying cell is
one or more whorls of membranes, sometimes interspersed with fragments of
electron-dense material, within vacuoles in the hypodermis.
DISCUSSION
We have described ultrastructural changes occurring in programmed cell
death in the developing nerve cord of C. elegans. The morphological sequence of
Figs. 9-12. Sections going posteriorly through Pll.aap. Condensed membranebounded fragments (F) of the dying cell are enclosed within an arm of hypodermal
cytoplasm (arrows), though continuity of this arm with the main body of the
hypodermal cell is not evident from these sections, x 23000.
98
A. M. G. ROBERTSON AND J. N. THOMSON
events implies that the hypodermis recognizes and surrounds the daughter cell
programmed to die almost immediately after birth of such cells at mitosis, from
the time the parent cell is in telophase and thereafter; either one or several
parallel arms of hypodermis extend around the dying cell and any membranebounded fragments which are formed. Ingestion then occurs until all that
remains within the hypodermis is a collection of membranous whorls interspersed with condensed chromatin-like remnants. The surviving daughter cell
shows no such recognition by the hypodermis and after division has normal
ultrastructure. In transverse sections through the whole nematode, membranous
whorls are also seen within the lateral hypodermis; these could be remnants
from other dying cells. It seems likely therefore that the hypodermis is specialized
in phagocytic activity, in concordance with published descriptions of its content
of esterase and acid phosphatase and its high metabolic activity (Bird, 1971).
Certain ultrastructural features observed in cell death in the nematode are
common to apoptosis (Kerr et al. 1972; Wyllie et al. 1980) the characteristic
mode of programmed cell death so far described in living tissues in mammalian,
avian, amphibian, reptilian and insect species studied; such features include
initial condensation of cytoplasm, nuclear chromatin aggregation, and fragmentation of the dying cell into membrane-bound fragments and the early
recognition by neighbouring phagocytic cells. Other morphological features of
cell death in the nematode, such as whorling of internal and plasma membranes
and autophagic vacuolation are atypical of apoptosis, in which there is also a
more pronounced degree of chromatin condensation. It is perhaps relevant
that cell deletion in the normal adult planarian (Bowen & Ryder, 1974) also
involves membrane whorling in dying cells though to a lesser extent than in the
nematode system; it is thus probable that lower invertebrates show a pattern of
programmed cell death differing in detail from that described in living tissues in
higher animals.
Neuronal cell death has been observed in other systems, for example with
normally degenerating cervical motor neurons and induced cell death in
peripherally deprived lumbosacral neurons in the embryonic chick spinal cord
(O'Connor & Wyttenbach, 1974) and embryonic neuron cell death in peripherally-deprived chick ciliary ganglia (Pilar & Landmesser, 1976). In these
systems, neurons appear to compete for synaptic sites on peripheral target
organs; those that fail to make synapses subsequently die. This, however, cannot
be the selection mechanism involved in the nematode as the dying cell does not
appear to form a nerve process. Thus the trigger of programmed cell deletion
in the developing ventral nerve cord of C. elegans has still to be determined. It
may involve contacts with hypodermal cells oi interneurons that touch the cell
body directly, or may be an intrinsic property of the deleted daughter cell which
is specified earlier in development.
Cell death in C. elegans
99
A.M.G.R. was supported by a grant from the Cancer Research Campaign to Professor
Sir Alastair Currie. We thank Dr J. Sulston for advice, Dr R. Horvitz and Dr A. Wyllie for
helpful discussion, and Mr S. Mackenzie for technical assistance. Transmission electron
micrographs were taken in the MRC Clinical and Population Cytogenetics Unit, Western
General Hospital, Edinburgh, and we acknowledge the help of Professor H. J. Evans and
Mr A. Ross.
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(Received 11 May 1981, revised 4 September 1981)