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Development 108, 83-96 (1990)
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Spatial and temporal patterns of neurogenesis in the embryo of the locust
(Schistocerca gregaria)
D. SHEPHERD and C. M. BATE
Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK
Summary
Embryonic neurogenesis in the ventral nerve cord of the
locust Schistocerca gregaria was studied using toluidine
blue (TB) staining and birthdating of cells by incorporation of bromodeoxyuridine (BUdR). In the thorax, the
neuroblasts (NBs) start dividing at the 28 % stage and
neurogenesis continues until 90%. In the abdomen,
neurogenesis starts at about 30 % and continues until
70 %. Every NB appears to have its own fixed period of
division before disappearing. Thus a specific spatial and
temporal pattern of NB degeneration can be seen in
every segment. This pattern is identical in each of the
three thoracic ganglia. We have traced the fate of each of
the original complement of NBs in the mesothoracic
neuromere and specified the stage of development at
which each NB ends its lineage and disappears. The
abdominal segments A2 to A7 share an identical pattern
of NB death, which is not comparable to the thoracic
pattern. The progress of neurogenesis is marked by a
gradual decrease in the number of NBs, with NBs in the
thoracic ganglia persisting longer than their abdominal
homologues. The differences between the thoracic and
abdominal NBs are also reflected in the rates at which
they divide, thoracic NBs dividing at almost twice the
rate of the abdominal NBs.
Introduction
later stages, neuron identity may not be so rigidly
specified. Studies of the later divisions of the NBs have
not been undertaken, because of the practical difficulties involved in the identification of individual NBs and
their progeny. Beyond the earliest divisions of the NBs,
the number of NBs that can be distinguished decreases
and the increasing number of neurons makes following
the progeny of individual NBs impossible.
Here we describe methods that have allowed NBs to
be visualised throughout embryonic development and
provide a detailed description of the entire process of
embryonic neurogenesis from the first appearance of
the NBs to the last division of the last NB. More
importantly, we have been able to trace the complete
life-history of each of the original complement of NBs
in the mesothoracic neuromere. With this information,
we can now individually identify all of the NBs present
in the mesothoracic ganglion at any particular stage of
development. The consequences of this are that we
have made the later stages of neurogenesis accessible to
a level of investigation not previously possible.
During neurogenesis in insects, the neurons in each
segmental ganglion are produced by a segmentally
repeated and stereotyped set of neuronal precursor
cells (neuroblasts NBs) and midline precursor cells
(Bate, 1976; Bate and Grunewald, 1981; Doe and
Goodman 1985a). Each NB divides asymmetrically and
repeatedly to produce chains of smaller ganglion
mother cells (GMCs) which divide once more to produce a pair of sibling neurones (Bate, 1976). The
identity of these neurons is understood to be specified in
three stages: first, the identity of each NB is uniquely
specified by its position in the neural ectoderm to
produce a particular and invariant family of neurons;
second, the identity of a pair of neurons is specified by
the rank order of their parent GMC in the lineage
produced by the NB and, third, an interaction between
sibling neurons decides which of two fates each will
adopt (Doe and Goodman, 1985a; 1985£>; Doe et al.
1985; Kuwada and Goodman, 1985). This model is
based almost entirely on observations of the earliest
stages of neurogenesis, from the enlargement of the
NBs in the neural ectoderm to the production of just the
first three or four GMCs. Some NBs however, produce
as many as 50 GMCs and the model has been extrapolated to cover the entire period of embryonic neurogenesis. Whether this model still applies at the later stages
is not known, and it is certainly possible, that at these
Key words: neurogenesis, insect, embryo, neuroblast.
Materials and methods
Embryos of Schistocerca gregaria (Forskal) from a laboratory
colony were used for all the work described here. The eggs
were laid in moist sand and stored in sand at a constant 30°C.
Eggs for experiments were removed from the sand and
84
D. Shepherd and C. M. Bate
cultured on moist filter paper in Petri dishes, at 30°C.
Embryos were staged according to the timetable of Bentley et
al. (1979).
Toluidine blue staining
Toluidine blue (Raymond Lamb) was used to stain living
nervous systems according to the recipe of Altman and Bell
(1973). Ventral nerve cords were incubated in the stain for
15-30 min at room temperature, then fixed and destained in
Bodian's fixative, dehydrated, cleared in xylene and mounted
in Canada balsam.
BUdR labelling
DNA synthesis was monitored using the incorporation of the
substituted nucleotide, 5-bromodeoxyuridine (BUdR), revealed immunocytochemically with a monoclonal antibody
against BUdR (Gratzner, 1982). This method has already
been used to study neurogenesis in the CNS of larvae of
Drosophila melanogaster (Truman and Bate, 1988). BUdR
(Sigma) was introduced into embryos by injecting a small
quantity (<l/i\) of 10mM-BUdR in embryo saline directly
into the embryo in ovo, with a glass micropipette. To assist
injection and to permit accurate staging of the embryos, the
eggs were dechorionated in bleach for 30-60 s and washed in
clean saline. Various concentrations of BUdR were tried;
concentrations less than 10 mM gave poor and variable labelling and concentrations higher than 10 mM were not used.
With lOmM-BUdR, labelling was strong and embryos developed normally and hatched. Therefore this concentration was
used in all experiments presented here.
Following injection, embryos were incubated on moist filter
paper at 30°C until required. In general, two protocols were
used. In the first, embryos were injected and incubated for
just 24 h and then processed. In the second, embryos were left
after injection to hatch and processed on the first day as 1st
instar larvae.
Nervous systems labelled with BUdR were either fixed in
situ (25-40% stage embryos) or dissected free prior to
fixation (40% to hatchling stage). Fixation was for 30 min in
Carnoy's fixative. Ganglia were then rehydrated and stored in
phosphate-buffered saline (PBS) with 0.3% Triton X-100
(PBS-TX). Prior to immunocytochemistry the preparations
were treated with 4N-HC1 in PBS-TX (1 to 1) for 30 min.
After thorough washing in at least six changes of PBS-TX, the
tissue was incubated in 10 % goat serum for at least 1 h at 4°C,
followed by incubation in a 1:200 dilution of the anti-BUdR
antiserum (Becton-Dickinson) in PBS-TX for 48 h at 4°C with
agitation. After the primary antiserum, the tissue was washed
for 8h with at least 8 changes of PBS-TX with agitation. The
tissue was then exposed to a rabbit anti-mouse IgG antibody
conjugated to peroxidase (1:250 in PBS-TX) for a further 48 h
at 4°C with agitation. The peroxidase was visualised according to the method of Watson and Burrows (1981). After the
reaction, the tissue was dehydrated, cleared in xylene and
mounted in Canada balsam. All the materials were examined
using x25, x40 and x63 oil immersion objectives on a Zeiss
microscope fitted with Nomarski optics.
Results
A study of the later stages of neurogenesis in the locust
requires that the NBs and their mitotic activity be
monitored throughout the full course of embryonic
development in every segment. At the early stages of
development (25-40% stage), it is possible to identify
all the NBs and observe their divisions in living embryos
using Nomarski interference contrast optics. During the
later stages of development, the growth of the nervous
system caused by the addition of more neurons and
their enlargement as they differentiate means that the
original NBs are displaced and become relatively small
making the resolution of individual NBs difficult. In the
absence of a NB specific stain, we cannot directly
observe neurogenesis beyond the earliest divisions.
Toluidine blue staining of NBs
Toluidine blue staining is a relatively selective stain for
NBs and Fig. 1 shows a series of TB stains of the
mesothoracic ganglion at different stages of embryogenesis. At the earliest stage (about 35 %), the NBs are
visible as large, ventrally located cells with large nuclei
and darkly staining cytoplasm (Fig. 1A). In addition to
the NBs, TB staining shows the early neuronal progeny
of the NBs and the intervening sheath cells. At this
stage, the NBs are quite distinct with an almost complete array visible in the mesothorax. By 50% of
development, the number of neurons is greatly
increased but NBs are still evident although relatively
smaller and spatially dispersed (Fig. IB). It is also
apparent by this stage that there are fewer NBs suggesting that some of them have completed their lineages
and have already degenerated. By 70 %, the number of
NBs still visible is smaller (approx. 30), and these
remaining NBs can be smaller than the surrounding
neurons (which have now enlarged) and recognisable
only by their shape and the associated chain of progeny
(Fig. 1C). It is still possible to distinguish NBs until
about the 85 % stage, but beyond this stage it is no
longer possible to identify them in any of the ganglia of
the ventral nerve cord.
Tracing NB mitotic activity with BUdR
Using TB we have been able to distinguish NBs in the
nervous system throughout most of embryonic development. It is possible, however, that the TB does not
reveal all the NBs in each ganglion, particularly in the
later stages of development when the apparent number
of NBs is low and they are small in size relative to their
rapidly enlarging progeny. We therefore used the incorporation of BUdR to reveal the presence of NBs by
their mitotic activity. For these experiments, embryos
were staged, injected with BUdR and allowed to
continue their development for a further 24 h before the
preparations were fixed and processed immunocytochemically with an antibody raised against BUdR. In
such preparations, clusters of small cells are labelled
(Fig. 2A), with labelling restricted to the nuclei of the
cells, each cluster contains a single large NB, several
(usually 3) GMCs and a variable number of neurons
(Fig. 2B). All the cells in a labelled cluster are the
progeny of the NB and each cluster therefore represents an active NB. Every NB that can be recognised
is associated with such a cluster of labelled cells. In
positively stained preparations, we never saw a NB that
was not associated with a cluster of labelled cells,
although occasionally we saw clusters of labelled neur-
Neurogenesis in insect embryos
85
Fig. 1. Whole mounts of the embryonic mesothoracic
ganglion stained with toluidine blue to highlight NBs at 3
different developmental stages. (A) 35%, NBs are clearly
visible as large cells with darkly staining cytoplasm (arrows)
Calibration 50 fan. (B) 50%, There are now many more
neurons evident but NBs are still clearly visible as large
darkly staining cells (arrows) Calibration 45jim. (C) 70%, By
this stage many neurons have enlarged (arrowheads) and
NBs (arrows) are no longer easily recognised. Calibration
20 nm.
onal cells that were not associated with a NB. Since the
timing and location of these clusters coincide with the
disappearance of NBs in TB-stained ganglia, it is likely
that these cells represent the final progeny of the NBs
concerned.
Labelling with BUdR at various developmental
stages can reveal the presence of NBs. Fig. 3 presents
examples of the metathoracic ganglion after injection of
BUdR at different stages, 30% (Fig. 3A), 55%
(Fig. 3B) and 70% (Fig. 3C). In all cases, the NBs are
revealed by their incorporation of the BUdR. Thus the
incorporation of BUdR is a graphic way of revealing
NBs in the developing nervous system. Furthermore,
these preparations highlight the developmental changes
in the number and distribution of NBs in this ganglion.
The results obtained using the incorporation of
BUdR are entirely consistent with those observed with
the TB staining. TB staining of early nervous systems
(<70 %) reveal the same pattern of NBs as found using
BUdR. BUdR incorporation is however, a more reliable and repeatable method and at later stages of
development revealed NBs not seen using the TB.
The temporal pattern of neurogenesis
Onset of neurogenesis
The earliest signs of mitotic activity in NBs can be seen
at 25 %, when NBs can be identified in every segment.
At this stage, however, not all the NBs are undergoing
DNA synthesis and cell division. There is an anteriorto-posterior gradient of neurogenic activity. Thus as
shown in Fig. 4, it is clear that, although most of the
NBs of abdominal segments 1, 2 and 3 are actively
dividing, in the more posterior segments there is less
86
D. Shepherd and C. M. Bate
evidence of division. This is reflected in two ways: first,
there are fewer NBs showing signs of BUdR incorporation and second, the labelled lineages produced by the
active NBs during a pulse appear to be smaller. Eventually, all the NBs start dividing but there is a slight
Fig. 2. (A) A whole mount of a BUdR labelled
mesothoracic ganglion showing the distinct clusters of
labelled cells generated by the NBs (the framed area is
shown enlarged in B). (B) At higher magnification each
cluster can be seen to include a single large NB, several
intermediate size GMCs and several small neurons (n). The
cells in the cluster are all the progeny of the associated NB.
The identities of the NBs are marked. Calibration A. 55/OTI
and B. 20/im.
anterior-to-posterior delay and, as development proceeds, progressively more posterior cells commence
DNA synthesis. Thus, by 30%, all NBs in every
segment can be seen to be synthesising DNA and
dividing. At this time, a clear pattern of NBs can be
recognised in every segment (Figs 5 and 6).
45-50%
At the 45-50 % stage, actively dividing NBs can be seen
in every segmental ganglion. The NBs no longer form
the tightly packed array typical of the earlier stages but
are becoming spatially separated (Figs 5 and 6). It is
also apparent that many NBs have produced their
entire lineages and have already degenerated. It is
assumed that the disappearance of a NB indicates that it
has degenerated. From our observations we have seen
that many NBs show clear signs of cell death (Bate,
1976) prior to their disappearance but, since we cannot
provide proof of the degeneration of all NBs, it is
possible that some NBs just cease DNA synthesis and
reduce in size. The losses are most obvious in the
abdominal segments where over 50 % of the NBs have
already disappeared (Table 1). In the thoracic ganglia,
they are fewer, with only 14 of the original complement
of NBs missing (Table 1).
The pattern of NB disappearance in each ganglion is
highly invariant and suggests that each NB has its own
specific timetable of development, that the active life of
each NB is uniquely determined and that it degenerates
at approximately the same stage of development in
every individual. Thus it is possible to trace the fates of
individual NBs and specify the point of degeneration.
For instance, NBs 6-3 and 7-3 are the first 2 NBs to
degenerate in the mesothorax and, by the 45% stage,
both are missing. It is possible to recognise the pattern
of NB division and degeneration in all ganglia and so
compare the patterns of NB death in different ganglia.
Thus, Fig. 5 shows a camera-lucida drawing of the
patterns of NB activity in the three thoracic ganglia of a
50 % embryo, a comparable pattern of NBs can be seen
in all three ganglia, with the exception of the anterior
median NB, which is found only in the prothorax. The
same can be done for the abdominal ganglia (Fig. 6); in
this case, all of the unfused abdominal ganglia (Al to
Table 1. The number of NBs visible in each segmental neuromere at various stage of development
(mean±standard deviation)
Stage
Ganglion
PRO
MESO
META
1
2
3
Total
ABD4
5
6
7
TAG
28%
35%
42%
59.6±0.8
59.0±0.0
58.7±0.5
58.0±0.0
56.011.6
54.511.0
50.014.5
53.812.3
53.412.2
44.514.4
42.013.9
37.312.5
177.3
45.514.0
47.312.9
46.312.5
50.112.1
142.314.0
47.512.1
50.011.4
49.012.0
31.012.0
23.811.1
23.511.0
126.1
27.010.0
29.010.1
30.010.2
31.010.1
109.0118.4
227
56.511.0
56.711.2
57.411.2
58.111.1
184.015.6
50%
44.311.9
47.512.1
43.812.5
22.011.2
16.313.2
14.513.0
93.9
21.312.1
23.312.1
23.712.3
25.010.0
67.0118.2
56%
63%
70%
77%
84%
91%
100%
40.014.8
37.412.2
38.012.1
12.014.4
7.513.4
7.513.4
65.0
9.613.4
8.010.5
9.010.4
13.616.7
42.512.2
32.612.8
33.412.0
28.312.5
30.311.2
19.511.0
18.511.0
4.511 .0
4.310 .5
0
—
-
-
—
—
-
—
-
0
0
-
23.710.6
4.410 .9
0
0
0
0
0
0
0
0
0
0
0
0
0
10.415.5
6.513 .4
0
59.311.9
8.711.5
8.410.5
10.111.9
8.015.3
37.218.2
45.711.2
4.711.5
5.811.6
6.010.8
6.613.7
29.0110.4
0
—
—
0
0
0
0
0
0
Neurogenesis in insect embryos
87
-A1
.. 2-2-T3
+
/\1
\
- A2
— A3
A7) show an almost identical pattern of persistent NBs
at the 50% stage. It is, however, not possible to
demonstrate a complete homology between the abdominal and thoracic patterns, although there are
examples of individual homologies, thus NBs 6-3 and
7-3 are the first NBs to degenerate in the abdominal as
well as the thoracic ganglia.
Any comparison with the terminal abdominal
ganglion (TAG) is difficult, as the TAG is unique in
representing the fusion of at least 3 abdominal seg-
Fig. 3. Whole mounts of BUdR labelling of the
metathoracic ganglionic mass a 3 different stages of
development illustrating how the presence of NBs is
revealed by their incorporation of BUdR. The
identity of some of the more prominent NBs is
indicated. (A) 30%, an early stage when most NBs
are present and dividing. At this stage the
metathoracic neuromere (T3) is separate from the
abdominal neuromeres that will later fuse with it.
Calibration 45 /im. (B) 55%, all the persistent NBs
can be seen with their chains of progeny (by this
stage the 1st abdominal neuromere (Al) has fused
with the ganglion) Calibration 75jan. (C) 80%, at
this stage most NBs have completed their lineages
and disappeared. The remaining active NBs are
revealed by their labelled progeny. Some of which
are no
longer associated with a NB (arrows), these
cells represent the final progeny of their parent NB
(the first three abdominal neuromeres (A1-A3)
have now fused with the metathoracic neuromere).
Calibration 100 \m\.
ments. Even so, a segmentally repeated pattern of NBs
can be recognised in the anteriormost of its constituent
segments at the earliest stages of development (Fig. 6),
but, by the 50% stage, it is not possible to recognise
with confidence any homologies with the more anterior
ganglia.
70%
In each thoracic neuromere, about half of the original
NBs have now degenerated and there are about 30 NBs
D. Shepherd and C. M. Bate
Fig. 4. Whole mounts of BUdR labelling of the nervous system of a 27 % embryo showing the anterior to posterior gradient
in the onset of mitotic activity of the NBs. (A) In the meso- and metathoracic ganglia NBs are clearly visible and associated
with many of them are clusters of cells that have incorporated BUdR (arrows). (B) 2nd and 3rd abdominal ganglia of the
same preparation and less BUdR incorporation is evident. Numerous NBs show no signs of BUdR incorporation (arrows)
and the labelled lineages contain fewer cells than in the more anterior ganglia Calibration 50/un. The planes of focus of
these micrographs have been adjusted to show the maximum levels of labelling possible for each ganglion.
30%
40%
60%
70%
8TS*
PRO
Fig. S. Camera-lucida drawings of the ventral
view of the thoracic ganglia of the embryonic
nervous system showing the patterns of NBs
revealed by BUdR incorporation at various
stages of embryonic development. Each
example in the figure was chosen because it
reflected most accurately the pattern typically
seen at each stage.
Neurogenesis in Insect embryos
30%
40%
60%
70%
89
85%
A4 - A7
Fig. 6. Camera-lucida drawings of the ventral
view of the abdominal ganglia of the embryonic
nervous system showing the patterns of NBs
revealed by BUdR incorporation at various
stages of embryonic development. The patterns
of NBs seen in ganglia A4-A7 are represented
by the one ganglion, each ganglion showing
identical patterns with a slight temporal delay
from anterior to posterior. Each example in the
figure was chosen because it most accurately
represented the pattern typically seen at each
stage.
actively dividing (Fig. 5 and Table 1). As at the previous stages, an identical pattern of extant NBs can be
seen in each of the three thoracic ganglia.
In most of the abdominal segments, however, only
the final traces of neurogenesis can be seen with only 5
or 6 NBs remaining (Fig. 6 and Table 1) and by 75 %
even these NBs have disappeared. Nonetheless at 70 %,
it is still possible to recognise an identical pattern of
persistent NBs in each of the segments A2 to A7. On
the other hand, it is also apparent that the NB activity
of segment Al differs from its more posterior counterparts in that there are as many as 10 NBs still dividing in
the ganglion (Table 1). The TAG also shows an extended sequence of NB activity, where almost 30 NBs
persist and continue to divide (Table 1 and Fig. 6).
85%-100%
By 85%, signs of neurogenesis have ceased in all
ganglia apart from the 3 thoracic ganglia and the TAG.
The number of active NBs in these ganglia is however
small, 4 or 5 in the thoracic ganglia and 6 in the TAG
(Table 1). As before, an identical pattern of NB activity
can be seen in all three thoracic segments (Fig. 5), and
no homologies can be recognised in the TAG. During
the next 24-36 h, all persisting NBs will complete their
lineages and degenerate, so that by 95 % all neurogenic
activity has ceased in the ventral nerve cord.
Sexual dimorphism of the TAG
Using BUdR, Truman and Bate (1988) showed that, in
the terminal ganglia of Drosophila larvae, there was a
sexual dimorphism in the time course of neurogenesis.
This was such that, in males, neurogenesis was extended by more than 30h. With this in mind, we made
particular observations for evidence of such a dimorphism in the TAG of locusts. Although it was not
possible to determine the sex of embryos, we were
unable, however, to detect any differences in neurogenesis that could be attributed to the sex of the animal.
Tracing the fate of individual NBs
As shown above, there is a consistent pattern of NB
disappearance in each ganglion suggesting that each NB
has a unique life-history, completing its lineage and
disappearing at a specific stage of development. With
the level of detail provided by the incorporation of
BUdR, we have been able to trace each of the NBs in
the mesothoracic neuromere from the first divisions to
disappearance. Following individual identified NBs
from one stage to the next is relatively straight forward
and is not complicated by migration, the NBs remaining
relatively close to their original positions. The biggest
movements are made by NBs 4-1, 4-2 and 4-3 which,
although they start in a line perpendicular to the
anterior-posterior axis, move such that by 70% 4-2
and 4-3 are displaced posteriorly (Fig. 7). The only
other movements are such that the more laterally
placed NBs (e.g. 6-4, 7-4, 2-5 and 3-5) are displaced
dorsally to come to lie on the dorso-lateral edges of the
ganglion. These NBs are however still identifiable by
their position relative to the other NBs.
From this information we are now able to identify
unequivocally and make a map of all the NBs present in
the ganglion at all developmental stages (Figs 7 and 8).
The full life-histories of all the individual NBs is shown
graphically in Fig. 9.
A fixed stage is given for the disappearance of each
NB, but there are however, slight variations in the exact
time of disappearance and therefore the stage given for
the disappearance of a NB is the latest stage at which
that NB has been identified.
90
D. Shepherd and C. M. Bate
Fig. 7. Identification of individual NBs. Whole mounts of the embryonic mesothoracic ganglion showing lineages labelled
with BUdR at 4 different stages of development: (A) 35 %, (B) 55 %, (C) 70 %, (D) 85 %. At each stage the precise pattern
of dividing NBs can be seen, from such preparations we have been able to trace the fates of individual NBs throughout
embryonic development. The identities of the parent NB of certain lineages is indicated. Calibration (A) 35;im, (B),(C) and
(D)55j/m.
30%
40%
60%
70%
0
o ooo
OQCOO
o
I
o0o 0
0°
Fig. 8. Camera lucida drawings showing the changing pattern of NBs in the mesothoracic ganglion during embryogenesis.
The figure serves to illustrate the developmental progress of 10 of the original complement of NBs. The identitities of the
NBs are given according to their position in the original NB artay.
Neurogenesis in insect embryos
MNB7-47-37-27-16^46-36-26-15-65-55-45-35-25-1 §4-4S 4-3-
INJECTION
DIVISION 1
0
4
91
DIVISION 2
7
I 4-2*4-l3-53-43-33-23-12-52-42-32-2"
GMCCVCLE-3XNB
11.25 CELLS
LABELLED IN
4 TO 5
DIVISIONS.
2-1 •
1-21-1-
DIVISION 3
60
70
Stage (%)
Fig. 9. A schematic presentation of the individual life
histories of all the NBs of the mesothoracic ganglion. The
bar indicates the active life of each NB, commencing at the
stage at which the NB is first recognised and terminates at
the latest stage at which the NB was seen and presumably
ended its lineage.
Rates of proliferation
Neurogenesis was further denned by comparing the
rates at which NBs produce labelled progeny in different ganglia. Embryos were staged, injected with BUdR
and then cultured in ovo for the required time before
fixation and processing. Following injection, labelled
progeny could first be detected after 3h and consistently thereafter for the next 21 h. Thus in a 24 h pulse,
the labelled cells are those that have gone through at
least one S-phase during the previous 21 h. The outcome of this regime is the labelling of the NB, 2 or 3
GMCs and a variable number of neurons. The rate of
proliferation was determined by counting the number
of labelled nuclei (GMCs and neurons) associated with
each NB. This rate does not give an exact figure for the
rate of NB division since some of the labelled cells may
be the progeny of a preexisting GMC and labelled as a
consequence of the division of the GMC. Given the
sample of cells used in this study, it is unlikely that this
fact influences the comparison of the rates for different
NBs, but it exaggerates the absolute rate of NB
DIVISION 4
11
DIVISION 5
13
Fig. 10. Schematic presentation of the presumed pattern of
BUdR incorporation in an actively dividing NB lineage,
illustrating the number of BUdR labelled cells produced by
each round of cell division by the NB and GMCs. In
normal animals, each dividing NB is almost invariably
associated with 3 GMCs and a variable number of neurons
(N). From this it assumed that the cell cycle time of the NB
is 3 times that of the GMCs and that with each division of
the NB the oldest GMC also divides. At the time of
injection of BUdR none of the cells is labelled. Earliest
labelling is seen after 3h when label will accumulate in 2
cells, the NB and GMC1, as they enter S-phase prior to
dividing. After the first round of division 4 cells are labelled
(excluding the NB, in all cases the number of labelled cells
does not include the NB), the newly generated GMC4, the
two progeny of GMC1, which has divided, and GMC2 now
in S-phase. The next round of division results in 7 labelled
cells: the newly generated GMC5, GMC4 labelled by the
previous NB division, GMC3 unlabelled in the previous
round has entered S-phase and 2 neurons from GMC2.
After subsequent divisions the increase in cell numbers is
simple with 2 newly labelled cells appearing for each round
of division. The NB producing 1 new GMC and the oldest
GMC producing 2 neurons, a net gain of 2 cells. The effect
of this pattern of division is that just 5 divisions of a NB can
result in the immediate labelling of 13 cells.
division. A more realistic estimate of the rate of NB
division can be made if one considers the patterns of
division made by NBs and their GMCs (Fig. 10). From
this, we can see that it is possible for a lineage to contain
four labelled cells (in addition to the NB) after only one
92
D. Shepherd and C. M. Bate
NB division. Only one of these cells was produced by
that NB division, of the other cells two are produced by
a GMC that has divided and the other is a GMC that has
entered S-phase. With the same logic, a lineage of 11
labelled cells (plus a NB) can be produced after only 4
divisions of the NB (Fig. 10).
In this study, we determined the proliferation rate for
4 uniquely identified NBs in the mesothoracic ganglion
(2-1, 4-2, 4-3 and 7-1) at 6 stages of development.
Data from 3 of these NBs are presented graphically in
Fig. 11; from this it is clear that NBs 4-2 and 4-3 are
producing progeny at the same rate, approx. 11.5
cells/24 h. Taking into consideration the patterns of
division shown in Fig. 9, this suggests a rate of NB
division of between 4 and 5 divisions in 24 h. This rate
declines simultaneously for both NBs as they come to
the completion of their lineages. The third NB (NB
2-1) appears at the early stages to be dividing at a rate
comparable to the others (11.25 cells/24h at 50%)
44
51
58
65
72
79
86
Developmental stage (%)
Fig. 11. Summary of the number of cells produced by
individual NBs during a 24 hour pulse of BUdR at different
times in their developmental history. B=NB 2-1, V=NB
4-3, #=NB 4-2 of the mesothorax and +=Abdominal
NBs.
again suggesting an actual NB division rate of 4 to 5
divisions in 24 h (Fig. 10), but this rate declines as it
nears its demise (65 %), earlier than NBs 4-2 and 4-3.
Two other identified NBs in the same ganglion were
examined and show more or less the same maximal
rates of cell division, declining only as they come to the
ends of their lineages (Table 2). The data suggest that
NBs in the same ganglion divide at approximately the
same maximal rate.
Do all the NBs divide at the same rate or are there
segmental differences? To answer this question, we
looked at the rates of division in other ganglia. In both
the prothoracic and metathoracic ganglia, NBs 2-1 4-2
and 4-3 all divide at rates comparable to their mesothoracic homologues (Table 2). In the abdominal
ganglia, it was not possible to examine the division rates
of individual identified NBs and so the data averaged
for at least 5 different but unidentified NBs are presented. These data show that the abdominal NBs divide
at a slower rate of 6.5 cells/24h, a rate equivalent to
only 2 NB divisions in 24 h (Fig. 10), a rate much slower
than their thoracic counterparts (11.5 cells/24 h)
(Table 2, Fig. 11). Since it was not possible to identify
individual NBs, we cannot compare the rates of division
of individual NBs. The NBs of the TAG show a rate of
division similar to the rates of the other abdominal
ganglia (Table 2).
It is possible that death of GMCs or neurons shortly
after their last division could serve to underestimate
cycling times of NBs (especially in the abdominal
ganglia), but from our observations cell death was not
seen as a significant factor amongst the labelled lineages
examined and we feel that it does not lead to a serious
underestimation of the rates of proliferation.
Other cell types
Although it is possible using BUdR incorporation to
identify the NBs and their progeny in the CNS unequivocally, neurons are not the only cell types labelled
by this procedure. There are at least 2 other classes of
proliferating cells in the embryonic CNS. Thefirstclass
of cells has large (30^m), flat nuclei that lie superficially
on the ganglia, connectives and nerve roots, forming
part of the extraganglionic sheath (Fig. 12). It seems
Table 2. The number of BUdR labelled cells generated by different NBs during a 24 hour pulse. Results are
presented for several different developmental stages (mean±standard deviation)
MESO
Stage
2-1
4-3
4-2
37%
11.2±0.9
n=8
10.8±1.6
n=6
9.910.6
n = 10
7.5±2.2
n=12
0
—
0
—
11.7±1.3
n=8
11.3±1.8
n=6
11.4±1.9
n=10
11.5±1.2
n=8
11.4±1.4
n=6
11.1±3.5
n=10
12.1±1.8
n=12
11.8±2.8
n=6
6.2±3.3
n=6
44%
51%
58%
65%
72%
12.4±2.9
n=12
11.5±1.9
tr=6
6.3±3.1
n=6
PRO
4-3
META
4-3
inn
?
TAG
?
_
—
11.3±2.1
n=>6
12.4±2.4
n=10
11.8±2.3
n = 12
11.5±1.3
n=6
2.8±1.9
n=6
11.3±1.7
n=4
11.0±1.2
n=4
11.2±1.5
n=6
11.8±1.0
n=4
6.3±2.2
n=4
6.0±2.5
n=25
6.3±1.0
n=8
6.8±2.0
n=18
5.6±1.5
n=23
4.7±1.8
n=6
5.9±1.4
n=16
5.9±1.6
n=17
6.4±1.7
n=17
0
—
5.8±1.8
n=16
6.4±1.7
n=19
6.2±2.4
n=18
Neurogenesis in insect embryos
93
Fig. 12. Other cell types in the nervous
system revealed by BUdR incorporation.
(A) Small irregularly shaped nuclei that lie
between the neuronal cell body layer and
the neuropile. This class of cells also
continue into the connectives and nerve
roots. Calibration 60 ^m. (B) and (C) A
second class of cells labelled has large flat
nuclei which lie superficially and appear to
form part of the perineurial sheath.
Calibration (B) 65 fm and (C) 40f«n.
likely that these are the nuclei of perineurial cells
(Wigglesworth, 1960) which differentiate to form part
of the perineurial sheath. The second class has small
irregularly shaped nuclei and appear to form a layer
between the outer cortex of neuronal cell bodies and
the neuropile. This class appears to extend into the
connectives and nerve roots (Fig. 12). On the basis of
this distribution, it is likely that these are the nuclei of
glial cells associated with the neuropile and connectives
(Wigglesworth, 1960). Although both types of cell are
numerous and readily labelled, they pose no problem
for the identification of neurons since the size, shape
and distribution of their nuclei are quite distinct from
those of the neurons.
The labelling of these two classes of glial cell with
BUdR reveals an interesting facet of their development. From the data presented here, it is apparent that
the two classes develop with completely different time
courses (Fig. 13). The so-called neuropilar glial cells are
first detected as labelled nuclei in embryos labelled at
about the 35 % stage and labelled cells of this class can
be seen consistently and in profusion after injections of
BUdR at all developmental stages thereafter up to
about 80 %. After 80 %, no more labelling is seen until
after hatching. The perineurial cells on the other hand
label during a much narrower developmental window.
The earliest labelling is at 45 % when a few (<20)
labelled nuclei are seen in a small number of preparations. Labelling reaches a maximum by 60% when
all preparations have a heavily labelled perineurium.
Labelling of this cell type ceases by 70% (Fig. 13).
Discussion
In recent years, neurogenesis in insects has become a
94
D. Shepherd and C. M. Bate
made only for the mesothorax, the marked similarity of
the patterns of NB persistence in the pro and metathoracic ganglia make it highly probable that the fate of
the individual NBs are the same in all three thoracic
ganglia and therefore future work need not be restricted to the mesothorax.
a.
3?
30
37
44
51 58 65 72 79
Developmental stage (%)
86
93 100
Fig. 13. Graphical representation of the times at which the
non neural cell types appear to incorporate BUdR.
Neuropilar glia (closed circles) appear in the majority of
preparations as early 37 % and can be seen to be labelled in
virtually all specimens until 86%. By contrast the
perineurial type cells (open circles) arefirstseen in a few
preparations at 44% and labelling continues until 79 %.
model system for studying the mechanisms that underlie the specification of neuronal identity (Doe and
Goodman, 1985a; Doe and Goodman, 19856; Doe etal.
1985; Kuwada and Goodman, 1985). All of these
studies have, however, placed their emphasis on the
earliest stages of neurogenesis; the determination of
NB identity and specification of the identities of the first
6 to 8 neurons produced by a NB. This restriction has
been enforced primarily by the difficulties encountered
in looking at and understanding the later divisions of
the NBs. With this work we have described a technique
that has overcome this restriction and has provided a
means of visualising and identifying NBs at all stages of
embryogenesis. Using this method we have been able
to: (1) describe the full timecourse and pattern of
embryonic neurogenesis in the ventral nerve cord, (2)
describe segmental differences in the timing and pattern
of neurogenesis and (3) trace the fate of all of the
original complement of NBs in the mesothoracic neuromere and make a complete record of the stage at which
each NB ends its lineage.
As a means of extending our knowledge of the later
stages of neurogenesis, perhaps the most valuable
product of this work has been the tracing of individual
NBs through development. Until this was done the
major limitation to tracing neural lineages in the locust
embryo was the inability to confidently identify NBs
beyond 50 %. Using the information that we now have
at our disposal, this is no longer a problem. It is quite
possible to look at the mesothoracic ganglion of living
embryos under Nomarski optics and identify unequivocally each NB. Coupling this with the conventional
techniques of intracellular recording and dye injection,
we can now examine the physiological and morphological properties of neurons produced by the later divisions of identified NBs. Moreover it is possible to trace
the complete lineage of identified NBs. This means that
the whole period of neurogenesis is now open to a level
of analysis once only possible at the earliest stages of
development.
Although a detailed examination of NB fate was
Spatial and temporal pattern of neurogenesis
At the onset of neurogenesis each of the presumptive
segmental ganglia has a more or less identical complement of NBs (Bate, 1976; Doe and Goodman, 1985a),
which are responsible for the production of virtually all
the central neurons in each ganglion. The ganglia
produced by these stereotyped arrays are, however, not
identical and, therefore, each array of NBs is capable of
producing not only the neurons of the simple abdominal
ganglia but also the neurons of the larger and more
complex thoracic ganglia. The mechanisms by which
this is achieved are not clear, although cell death and
variations in the lifespan of NBs are known to be
important (Booker and Truman, 1987a, 19876). Using
BUdR and TB, we have been able to trace neurogenic
activity in each segment from its onset to the final
divisions of the last NB. Our data suggest that in the
locust at least two factors can contribute to this difference in the length of the lineages produced by NBs in
different ganglia: that is, segmental differences in the
life time of individual NBs, and in their rates of
division.
The time course of neurogenesis
The time course of neurogenesis divides roughly into a
thoracic pattern and an abdominal pattern. The first
signs of division in the thoracic NBs occur at about
25%, but not until about 28% do all thoracic NBs
appear to be actively dividing. Neurogenesis then
continues unabated in all 3 thoracic ganglia until shortly
before hatching (92%). The persistence of neurogenesis to this late stage is quite remarkable since it shows
that neurons are being generated to within 20 h of the
embryo hatching. Studies of the progeny of the median
NB of the metathoracic ganglion show that the development of a neuron from axon outgrowth to the onset of
electrical excitability can take up to 25 % of development (i.e. 3.5 days in Schistocerca) (Goodman and
Spitzer, 1979). With such a time course, it is unlikely
that the neurons produced by NBs at these late stages
could have fully differentiated before hatching and it
may be that they complete their differentiation during
larval life. The abdominal NBs (A2-A7), by contrast,
start division later and in an anterior-posterior sequence. Thus division in A2 begins at 28% as against
35 % in A7. Neurogenesis in these ganglia ceases at
70%, but once again in an anterior-posterior sequence. The NBs of Al and the TAG have unique
patterns of division. In Al the pattern is an intermediate one, division commencing at the same time as in the
thoracic NBs and continuing until 77%, (i.e. longer
than in the other abdominal ganglia but shorter than in
the thorax). The NBs of the TAG are among the last to
start dividing but dividing NBs persist until well after
Neurogenesis in insect embryos
they have disappeared in all other abdominal ganglia.
Neurogenesis in the TAG ends at the same time as in
the thorax.
Although the temporal sequence of neurogenesis we
describe suggests a continuous sequence of NB divisions, this does not mean that all of the NBs are active
throughout the entire period. There is in fact a continual loss of NBs from all segments throughout neurogenesis. It is clear that the thoracic ganglia differ from
their abdominal counterparts not only in the temporal
pattern of neurogenesis but also in the rate at which
NBs complete their lineages and degenerate. For
example at the 50% stage, only 15 of the original 61
NBs in each thoracic ganglion have degenerated as
against 40 in each of the abdominal ganglia (A2-A7).
In this way, neurogenesis is not only extended in the
thoracic ganglia, but more NBs persist to the later
stages than in the abdominal ganglia. This is very
similar to the pattern of neurogenesis seen in the larval
nervous system of holometabolous insects such as
Drosophila (Truman and Bate, 1988) and Manduca
(Booker and Truman, 1987a). In Schistocerca, however, neurogenesis is completed in the embryo and does
not extend into larval life.
The differences between the ganglia are not reflected
solely in the timing of onset and termination of neurogenic activity but also in the spatial pattern of NB
degeneration. Each NB has a specific lifespan, and
divides according to a particular programme to produce
its progeny and then degenerates (there is no evidence
to suggest that this is an exact number of divisions).
Thus cell death is a consistent and predictable event for
any NB and there is a distinct sequence of NB deaths in
each segment. Each of the 3 thoracic ganglia have
identical spatial patterns of NBs at all developmental
stages (with the exception of the anterior median NB in
the prothorax) suggesting that homologous NBs in each
segment undergo exactly the same patterns of division
and degenerate at the same stage in each of the 3
ganglia. Like the thoracic ganglia, an identical pattern
of NBs can be seen in the abdominal segments (A2-A7)
at all developmental stages (the A9-A11 possess an
anterior median NB) again suggesting that segmentally
homologous NBs share the same pattern of mitotic
activity and degenerate at equivalent times in development. It is not possible, however, to see any homologies
with the sequence of NB death seen in the thoracic
ganglia. The first abdominal segment and the TAG are
different from the other ganglia. In Al, the rate of NB
death resembles that seen in the other abdominal
segments until about 55 % when several NBs survive
beyond their homologues in posterior segments. The
TAG is unique and cannot be compared with any other
ganglia and no homologies can be recognised.
All of these intersegmental differences indicate that
the complexity of, for example, the thoracic ganglia is in
part generated by the extended divisions of more NBs.
Similar extensions of neurogenesis in Al and the TAG
are also indicative of the specialisation of these ganglia;
e.g. Al becomes fused with the metathoracic neuromere and presumably contributes central neurons re-
95
sponsible for the control and processing of information
related to locomotion (Robertson and Pearson, 1984)
and ventilation (Hill-Venning, 1988). Similarly, the
TAG is specialised in being responsible for the control
of the genitalia and primary processing of information
from the sensory structures associated with the genitalia
(Thompson, 1986; K.J. Seymour pers. comm.). In
contrast to Drosophila where in the males neurogenesis
in the terminal ganglia is extended to later stages than in
females (Truman and Bate, 1988), there is no sexual
dimorphism apparent in the pattern of neurogenesis in
the terminal ganglia of the locust.
The rate of proliferation of NB lineages
These data tell us more about the development of the
segmental specialisations. If one considers the rates of
division of the NBs in the different segments, it can be
seen that not only do the thoracic NBs persist longer
and in larger numbers but also produce progeny at a
more rapid rate (11.5 cells day" 1 ), which is equivalent
to 4 or 5 NB divisions every 24 h, than their abdominal
homologues (6.5 cells day ), which equals a NB division rate of just 2 divisions in 24h. Therefore, the
complexity of the thoracic ganglia is doubly manifest.
Again these results show strong parallels with similar
data obtained for the larval NBs of Drosophila, which
have the same higher rate of division for thoracic NBs
(Truman and Bate, 1988). In Drosophila, the overall
rates of division are much higher than in the locust but
the ratio of thoracic to abdominal NB proliferation
rates is almost identical for both insects. Interestingly,
Al and TAG, which are of intermediate complexity in
terms of having an extended period of neurogenesis, do
not show a rate of proliferation significantly greater
than their counterparts in the simpler abdominal
ganglia and therefore their specialisations are due
primarily to the extension of their mitotic activity.
We would like to thank Drs Alfonso Martinez Arias, Gilles
Laurent and Helen Skaer for reading the manuscript. This
work was funded by the SERC(UK).
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{Accepted 10 October 1989)