Department of Biology and Graduate Program in Neuroscience, Emory University, Atlanta, GA 30322, USA
* Author for correspondence (e-mail: msiegler{at}biology.emory.edu)
Accepted 6 August 2002
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SUMMARY |
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Key words: Engrailed, MP, MNB, Lineage, Cell death, Asymmetry
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INTRODUCTION |
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The MNB lineage of grasshopper has provided a longstanding model for the development of large NB lineages containing differing neuronal types within the insect CNS. Perhaps more so than within any other NB lineage, individual neurons and small neuronal groups of like type have been broadly investigated, not only from a developmental perspective, but also in morphological, biochemical and behavioral studies of the adult. The present study focuses on the mechanisms by which the MNB gives rise to different neuronal types across the lineage.
The prevailing lineage model is a `sequential' one, whereby the ganglion
mother cell (GMC) divisions produce sibling pairs first of efferent neurons,
then pairs of interneurons, either local interneurons or intersegmental
interneurons (Goodman et al.,
1980) (for reviews, see
Burrows, 1996
;
Truman et al., 1993
). The
morphology of neurons in the MNB (or DUM) groups during embryogenesis and in
adulthood is consistent with this model
(Thompson and Siegler, 1991
;
Thompson and Siegler, 1993
).
However, En-positive neurons are produced from the MNB early in the lineage
(Condron and Zinn, 1994
;
Condron et al., 1994
).
Engrailed expression is specific to interneurons
(Siegler and Pankhaniya,
1997
), and we were thus led to question the validity of the
sequential model. We traced MNB and midline precursor (MP) lineages across
embryogenesis in grasshopper, with the aim of discovering the order in which
neurons of different types were produced, and what role En might have in this
process.
Our results confirm some earlier findings, but at the same time lead to a
significant revision in understanding of the MNB lineage and its progeny. We
show that, as in Drosophila, the MNB in grasshopper does not produce
midline glia, contrary to previous reports
(Condron and Zinn, 1994;
Condron et al., 1994
). More
importantly, we show that the MNB lineage is asymmetric in its production of
neuronal progeny. Throughout the lineage, each GMC generates two nonidentical
siblings: one En-positive and one En-negative neuron. A proportion of each
neuronal type dies during the course of embryogenesis, but death is most
pronounced among the En-negative efferent neurons. Thus, differential cell
death, rather than a timed lineage sequence that produces different neuronal
types, accounts for the significantly smaller number of efferent neurons
versus interneurons in the final neuronal populations.
We argue that the continued production of neurons of two essentially different types within a lineage, followed by selective death of some neurons, is normal across insect neuronal lineages. This provides a flexible and responsive mechanism whereby neuronal populations can be tailored to match segmental diversity across the insect body plan, without reconfiguring individual neuroblast lineages. A similar mechanism would likewise provide a ready means of matching neuronal populations in the CNS with changes in peripheral body form across the course of insect evolution, while not altering the central mechanism of neuronal production. This view is concordant with numerous findings across a diversity of organisms, where divisions of precursors yield progeny of asymmetric type, followed by selective cell death.
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MATERIALS AND METHODS |
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Antibodies
Primary antibodies were obtained from the following sources: anti-En/Inv
(MAb 4D9) and anti-BrdU (MAb G3G4) from the Developmental Studies Hybridoma
Bank; MAb 8B7 (unidentified cytoplasmic epitope) and anti-Lachesin from M.
Bastiani (U. Utah); and anti-Annulin (MAb 7H7) from D. Bentley (UC Berkeley).
Commercial antibodies were: anti-BrdU (CalTag); rabbit anti-HRP-fluorescein
isothiocyanate (FITC) #323-095-021 or lissamine rhodamine sulfonyl chloride
(LRSC) #123-085-021; goat anti-mouse-indodicarbocyanine (Cy5) #115-175-003 or
FITC #115-095-003; and goat anti rabbit-FITC #111-095-003 (Jackson
ImmunoResearch).
Immunohistochemistry
Standard protocols were used for immunohistochemistry. Tissue was blocked
in 5% normal goat serum (NGS) in saline, incubated in primary plus NGS
overnight at 4°C, washed and incubated with secondary antibody preadsorbed
against macerated fixed embryos. For triple-labeling with propidium iodide
(PI), anti-En (4D9) and HRP Ab, the steps after fixation were: wash, incubate
in 1 µg/ml PI for 2 hours at room temperature (RT), wash, block in NGS and
incubate in 1:1 MAb4D9 in saline plus NGS overnight at 4°C; wash, block in
NGS and incubate in goat anti-mouse Cy5 secondary for 2 hours at RT; wash,
block and incubate in rabbit anti-HRP-FITC overnight at 4°C, wash and
mount. For En/anti-HRP Ab double-labeling, PI was omitted. Tissue was mounted
in SlowFade or ProLong (Molecular Probes).
One concern with En/BrdU/anti-HRP Ab triple-labeling was that only same species primaries (mouse monoclonals) were available for En and BrdU detection, and both are nuclear labels. Thus, for En/BrdU/anti-HRP Ab triple-labeling, additional steps were included. After incubation in 1:1 4D9, block and Cy5 secondary incubation, tissue was again fixed in 3.6% paraformaldehyde for 1 hour. The next steps were: wash and incubate in 2.5 N HCl in saline for 30 minutes to cleave DNA; wash, block and incubate in anti-BrdU mouse MAb; block, wash and incubate in goat anti-mouse FITC secondary; wash and incubate in anti-HRP-LRSC; wash and mount. The second fixation eliminated cross-reactivity. In all preparations, scored independently by both investigators, all four of the possible combinations of BrdU (positive or negative) and En (positive or negative) were found, indicating that false positives and false negatives had been eliminated in the procedure.
Microscopy
Confocal microscopy used a BioRad 1024 laser scanning confocal microscope
and Zeiss compound microscope. Over 300 preparations were examined. A
z-series of images was collected using simultaneous or sequential
laser line scanning as appropriate for the fluorophore combination. For
triple-labeling with LRSC or PI, FITC and Cy5, sequential scanning was used to
avoid mixing of the LRSC (or PI) and FITC signal. Additionally in some cases,
one channel was set with a transmitted light detector to record a confocal DIC
image. Four sweeps were averaged for each step. Digital files were analyzed
frame-by-frame, each frame being one step in a z-series. Scoring of
cells for the presence or absence of a particular marker (e.g. BrdU, En, HRP)
was done independently for each detection channel.
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RESULTS |
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MPs 4-6 and MNB are separate neuronal progenitors
The progression of neuromere development is seen by labeling with an
anti-HRP antibody (HRP-labeling) (Fig.
1A). The antibody recognizes a carbohydrate epitope present in
many membrane proteins of insects (Snow et
al., 1987). Neuronal labeling is readily distinguished from
non-neuronal (glial) labeling. Neuronal labeling is distinctly punctate,
whereas glial labeling is diffuse and weakly above background. Scattered,
punctate HRP-labeling appears on developing neurons shortly after MP progeny
or GCs are produced by division. Within a 1% developmental interval, the label
is sufficiently strong to define the circumference of neuronal somata. As
growth cones, neurites and axons arise, they also express the HRP label. This
neuronal labeling persists, making it a robust marker of neuronal
phenotype.
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Embryonic segments have an anterior En-negative region and a posterior En-positive region (Fig. 1A). The MNB, three of the MPs (MP4, MP5 and MP6) and NBs of rows 6 and 7 arise from the En-positive region. By 25% neuromere development, MP4 is apparent near the posterior of the En-positive region (Fig. 1B,C). MP4 is larger in diameter, more dorsal and has more intense En expression than surrounding cells at the midline. At 25%, the incipient MP5 is just posterior to MP4, and by 26% MP5 is equal to MP4 in size and En expression (Fig. 1B-D). By 27%, MP4 has divided to form two progeny, both of which are initially En positive, and by 28% MP5 has likewise divided (Fig. 1E,F). By 28%, the two MP4 progeny show the strong, punctate HRP-labeling characteristic of neuronal identity, whereas in the two MP5 progeny HRP-labeling is barely detectable (Fig. 1F). By 29%, one of the two MP4 sibling neurons now has a conspicuously reduced level of En expression (Fig. 1G). MP6 and the MNB differentiate from 26% onwards. At 26% the incipient MP6 is just ventral to MP5, but by 28% comes to lie posterior to the two MP5 progeny, and by 29% has itself divided to form two progeny, both of which are also initially En-positive (Fig. 1D-G). Differentiation of the MNB from the neurectoderm has a time course similar to that of MP6, and at 28% lies next to MP6, but is somewhat more ventral and is associated with its glial cap cell (Fig. 1F).
By 31% neuromere development, MP6 has divided to produce two progeny (Fig. 2A). At this time, the MP5 progeny, as well as the earlier-differentiating MP4 progeny, show strong, punctate HRP-labeling, whereas in the two MP6 progeny HRP-labeling is yet barely detectable (Fig. 2A,B). Stereotypic changes occur in the relative positions of the MP progeny. Initially, pairs of siblings lie side by side (e.g. Fig. 1E,F and Fig. 2A), but as neuronal differentiation proceeds, one of each pair comes to lie dorsal to the other (Fig. 2A,B,D). In addition by 31%, the MNB has produced at least three GMCs, which lie in a stack above it, the first born being the most dorsal (Fig. 2I). Glial nuclei are already in place at the circumference of the MNB (Fig. 2C). Sometime after 31%, and occasionally as late as 32%, the first GMC divides to produce the first pair of GCs. In the example shown in Fig. 2A,B, division of the first GMC is under way, but not complete. The incipient GC siblings have strong Lachesin surrounds, particularly at the central face between them, and the condensed chromosomes are positioned at their opposite poles.
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En expression is regulated during maturation of MP4-6 progeny and in
MNB and its progeny
After each MP4-6 division, the two progeny initially have equally strong En
expression, but this is maintained in only one sibling. A difference in En
expression in the two MP4 progeny, born 27%, is just detectable at 28%
(Fig. 1F) and is pronounced by
31% (compare 4d, 4v in Fig.
2E,F). By 32%, one sibling has no detectable level of En, whereas
the other maintains a high En level (compare 4d, 4v in
Fig. 2G,H). The two MP4 progeny
thus differentiate as one En-positive and one En-negative neuron between 27%
and 32%, i.e. during a 4-5% developmental interval. The sibling pairs from MP5
and MP6 differentiate likewise, over similar intervals
(Fig. 2E-H). This process is
completed successively later in the MP5 and the MP6 siblings, reflecting the
later times of MP5 and MP6 division (see
Fig. 4A). Downregulation of En
expression in one neuron of each pair occurs as the somata shift from a
side-by-side, to a dorsoventral orientation. The sibling that maintains a high
level of En expression invariably comes to lie more ventrally and the sibling
that downregulates En expression, more dorsally. A difference in orientation
is evident before differentiation of the En-positive and En-negative fates is
complete (Fig. 2E,F).
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The MNB is En-positive initially (Fig. 1F). However, En expression is never as strong as it is in MPs 4-6, and by 29% the MNB has a background level of En (31% is shown in Fig. 2F). By contrast, MNB GMCs become En positive as they mature. The GMCs form an ordered stack or column, with the newest GMC adjacent to the MNB. When GMCs first arise from the MNB they are En-negative, but gradually become strongly En-positive, expression being highest in the oldest GMC (e.g. GMC1 in Fig. 2E). En upregulation across the maturing GMCs is seen throughout development, as shown in a 36% neuromere (Fig. 2I-K) and a 55% neuromere (Fig. 2L).
The MPs 4-6 and the MNB differentiate within a glial surround, which arises early in midline development. At 27.5%, prior to differentiation of the MNB, glia surround the cluster containing the two MP4 progeny, MP5 and MP6 (Fig. 2M). Slightly later, the MNB and its earliest progeny are also encompassed by glial cells (31.5% is shown in Fig. 2N; Fig. 3F). En-positive glial nuclei are evident at the circumference of the MNB, for example by the time it produces its first GMCs (shown at 32% and 33% in Fig. 2F-H). The glial surround persists throughout development, as shown, for example, in a 43% neuromere (Fig. 2O).
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Development of type-specific midline neurite bundles
The midline fiber tract comprises two separate bundles, starting with the
outgrowth of growth cones from the two MP4 progeny. By 31-32%, only
shortly after MP6 divides, both of the MP4 neurons have well-established
neurites and growth cones oriented anteriorly
(Fig. 3A-C). The two neurites
can be traced individually to the cell bodies of the two MP4 progeny, one of
which (4v) is more ventral (and remains En positive), and one of which (4d) is
more dorsal (and becomes En negative) (Fig.
3B,C). The neurite of the En-positive sibling (4v) branches first,
at the posterior midline. In 31-32% neuromeres, HRP-labeled filopodia of the
4v sibling contact growth cones of lateral neurons that are growing towards
the midline (Fig. 3A,C). This
is the first indication of the ventral region of the midline fiber tract, and
of the incipient posterior commissure (PC). The ventral region of the midline
fiber tract eventually includes all En-positive neuronal progeny of the MPs
4-6, and of the MNB. Thus, the En-positive MP4 sibling pioneers the PC, as
well as a distinct midline bundle. A few percent later, the 4d sibling is the
first midline neuron to reach the anterior commissure (AC) and then to branch
bilaterally. It thus pioneers the dorsal region of the midline tract, which
eventually includes all En-negative progeny of the MPs 4-6, and of the MNB.
The incipient AC is already delineated at the midline
(Fig. 3A), so the 4d sibling
does not pioneer the AC.
We found that anti-HRP and MAb 8B7 labeling reveal different neuronal
features. MAb 8B7 was used extensively in earlier descriptions of the MNB
group (Condron and Zinn, 1994;
Condron et al., 1994
), and thus
provides important points of reference. The 8B7-label reveals the core of
individual neurites, but strong, well-delineated 8B7-label is transient on
somata. Labeling of somata by 8B7 is faint in the first stages of neuronal
differentiation, at the time when punctate HRP-label first appears, and is
highest during the early stages of neurite extension. For example, 8B7-label
is stronger on the somata of MP progeny that have neurites (4d, 4v) than on
younger progeny (e.g. 5d) (Fig.
3A,B), and is pronounced on the soma of a lateral neuron that also
has strong 8B7-labeling of its relatively undeveloped neurite
(Fig. 3D). Neurons with longer
primary neurites or axons, i.e. having extended a distance at least two to
three times the measure of the somata diameter, have only faint 8B7-label on
their somata, but sustain the label on neurites and axons
(Fig. 3E, lower panel). By
contrast, HRP-label is apparent throughout these neurons
(Fig. 3E, upper panel).
The restricted nature of the 8B7-label was useful in tracing further development at the midline (Fig. 3G-N). By 35-36% the six MP 4-6 progeny have mature levels of En expression. The three En-negative somata have assumed relatively dorsal positions and the MP4 sibling (4d), at least, has bilateral branches at the AC (Fig. 3G). The three En-positive somata remain relatively ventral, and two or three siblings typically have bilateral branches at the PC (Fig. 3H). The bundle containing the En-negative neurons is separate from that containing the En-positive neurons, and typically is immediately dorsal to it. Occasionally, however, the bundles are offset laterally, making their separate identities clear (as shown in a 38% neuromere, Fig. 3I,J). By 38%, all six MP neurons have branched in their respective commissures, and additional midline neuronal progeny have been contributed from the MNB lineage.
At later stages, the axons of the MP neurons could be traced past the edges
of their respective commissures (data not shown). Two of the En-negative
efferent neurons had bilateral branches that grow into a small anterior tract
that exits nerve 1 (N1). They appear to be equivalent to the two DUM1 efferent
neurons in the adult (Campbell et al.,
1995). The third En-negative efferent neuron had bilateral
branches that grew towards lateral nerves 3-5, and thus might be equivalent to
any one of several adult DUM neurons. The three En-positive interneurons had
bilateral branches that join an ascending longitudinal tract. They thus
prefigure the morphology of adult DUM interneurons
(Siegler and Pankhaniya, 1997
;
Thompson and Siegler, 1991
).
The six MP4-6 neurons remained closely associated with the MNB progeny
(Fig. 2G-K,
Fig. 3G-K). Thus, what is
commonly referred to as the `DUM group' or the `MNB group' of the adult also
includes the MP4-6 progeny.
The median nerve, which has a portion of its path along the midline, is
developing at the same time. At 38%, the peripheral and central portions of
the incipient median nerve have not yet joined. The neurons that form the
distal portion of the nerve grow inwards from the periphery, but have not yet
met at the midline (vertical arrows, Fig.
3L). The central region arises from paired neurons of bilateral
origin, including the spiracle motoneurons. Their axons have grown to the
midline, dorsal to the PC, and are extending posteriorly (oblique arrows,
Fig. 3L). A `Y' shape is thus
formed just posterior to the PC, which superficially resembles the bilateral
PC branching of interneurons from MP4-6s and the MNB
(Fig. 3M,N). The median nerve
is quite separate in development, however. By 43% the proximal and distal
portion of the median nerve have joined, and a portion of the median nerve
traverses the cluster of neuronal somata produced by the MNB (data not shown).
This morphology is evident also in adulthood
(Thompson and Siegler,
1991).
The two midline bundles were traced to their mature form (data not shown).
The bundle pioneered by 4v ultimately becomes the posterior DUM tract (PDT) of
the mature ganglion, whereas the bundle pioneered by 4d ultimately becomes the
superficial DUM tract (SDT) and/or deep DUM tract (DDT). The PDT comprises
En-positive DUM interneurons, and the SDT/DDT comprises the En-negative DUM
efferents (Thompson and Siegler,
1991).
The MNB lineage: each GMC division yields one En-positive and one
En-negative neuron
During the early development of the MNB lineages, up to 50% in T3 and 45%
in A1 and posterior, newly differentiated neurons included both En-positive
neurons (interneuronal fate) and En-negative neurons (efferent fate) in about
equal numbers. This outcome was not expected: by the sequential model, the
early part of the lineages should be composed of En-negative neurons alone. We
therefore hypothesized that, as in the MP4-6 lineages, neurons of two types
arise from each GMC division. We tested this hypothesis, first, by counting
En-positive and En-negative neurons throughout development and, second, by
labeling with BrdU to trace the birth of sibling neurons.
Evidence from population counts
En-positive and En-negative neurons increased in an essentially 1:1
fashion, through 45% embryogenesis. At 40%, neuronal counts (mean±s.d.)
were 9.0±1.3 (En-positive) versus 8.2±1.1 (En-negative) in T3,
and 6.8±3.7 (En-positive) versus 6.6±1.3 (En-negative) in A1. At
45%, counts were 17.0±3.0 (En-positive) versus 15.0±2.0
(En-negative) in T3, and 12.3±1.9 (En-positive) versus 11.2±1.3
(En-negative) in A1 (Fig.
4B,C). From 50% onwards, however, the numbers diverged
conspicuously, to yield midline populations containing many more En-positive
neurons than En-negative neurons. By 55% development, the populations have the
mature number of En-negative efferent neurons. The first embryonic molt, from
45% to 55% embryogenesis, yields substantial cell death, particularly in
abdominal neuromeres (Thompson and
Siegler, 1993). Counts after 45% therefore do not measure the
absolute numbers of the two neuronal types produced. However, taken together
with the BrdU data (below), these data allow a minimal estimate for the extent
of cell death among the neurons of efferent fate.
Evidence from BrdU labeling
To test our hypothesis directly that each GMC division gives rise to one
sibling with an En-negative efferent fate and one sibling with an En-positive
interneuronal fate, embryos were injected with BrdU. Forty-eight hours later,
embryos were triple-labeled to assess BrdU incorporation, neuronal
differentiation, and En expression (Fig.
5A,B). We sampled injection times across embryogenesis, but
focused on two: 35% and 55%. By about 35%, the first GCs are produced. By 55%,
the groups contain the mature number of En-negative (efferent) neurons, but
less than the mature number of En-positive neurons
(Fig. 4B,C). By the sequential
model, 55% would be well past the time that efferent neurons are produced.
Embryos injected at 35% matured to 43-44%, thus prior to the onset of neuronal
cell death. Embryos injected at 55% matured to 63-65%, at or shortly before
the time the MNB dies. For early and late intervals alike, we tallied
En-positive and En-negative neurons (HRP labeled) that were born after BrdU
injection (BrdU positive).
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En-positive neurons and En-negative neurons labeled with BrdU were essentially identical in number, during both intervals, irrespective of the total number of neurons labeled. For injection at 35%, the number of BrdU-labeled neurons (mean±s.d.) was 7.5±2.4 (En positive) versus 7.6±2.3 (En negative) (Fig. 4D). For injection at 55%, the number of BrdU-labeled neurons was 7.1±2.9 (En positive) versus 6.7±2.7 (En negative) (Fig. 4E). Counts made from a sampling of other time points, and including other neuromeres (e.g. T2 and A2), likewise showed that equal numbers of En-positive and En-negative neurons were born from the MNB lineages.
The numbers of BrdU-labeled En-negative versus En-positive neurons in individual preparations were strongly correlated. The coefficient of correlation was 0.99 for T3 and A1 together, and 0.99 and 0.98, respectively, for T3 and A1 separately, for injections at 35% (Fig. 4D). The coefficient of correlation was 0.88 for T3 and A1 together, and 0.93 and 0.90, respectively, for T3 and A1 separately, for injections at 55% (Fig. 4E). All labeled T3 and A1 neuromeres were included in these data, and confirmed the prediction that equal numbers of the two neuronal types would be labeled irrespective of the total number of BrdU-labeled neurons. The average number of BrdU-labeled neurons in total (En-positive plus En-negative) was similar for the two intervals, 15.5±4.6 and 13.8±5.4 (35% and 55% injections, respectively). The range in number of BrdU-labeled neurons was eight to 24 neurons and five to 24 neurons (35% and 55% injections, respectively). Some neuromeres counted had complete labeling of the lineage (the MNB and all GMCs and GCs having BrdU labeling), whereas others had less complete labeling (some GCs and/or GMCs did not have BrdU labeling). Incomplete labeling would have occurred if BrdU was diluted away from the injection site, for example.
For injections at 35% embryogenesis, we also counted midline neurons that were BrdU-negative after the 48 hour interval. The numbers of each type were (mean¶s.d.) 4.3±0.7 (En-positive) versus 4.3±0.7 (En-negative), or a total of 8.6±1.4 neurons. The sample comprised 24 neuromeres, 12 from T3, and 12 from A1. In 22 of the neuromeres, the counts of En-positive neurons/En-negative neurons respectively were 3/3, 4/4 or 5/5. In two cases, these in A1, values were 4/5, possibly reflecting the slightly later onset of HRP-label in the En-positive neurons. Three of the neuronal pairs could be identified from position and soma diameters as the MP4-6 progeny, whereas the additional one or two pairs are neurons produced by the MNB lineage.
Although each GMC division produces two neuronal progeny that are of unlike type, near the end of embryogenesis the midline groups contain greatly more En-positive neurons than En-negative neurons (Fig. 4B,C). These counts allow a minimal estimate of the extent of cell death, at least among En-negative progeny of efferent fate. In T3, counts at 85% embryogenesis show about 65 En-positive versus 19 En-negative neurons, or roughly 45 fewer En-negative neurons. In A1, counts at 85% embryogenesis show about 52 En-positive versus three En-negative neurons, or roughly 50 fewer En-negative neurons. From the disparity of numbers it is clear that cell death is crucial in shaping the mature populations. It can be inferred that the total number of progeny produced in the lineages are at least twice the number of surviving En-positive progeny. Thus, in T3 at least 35% (45/130) of the neurons, and in A1 about 50% (50/104) of the neurons produced in the MNB population die across embryonic development. This is an underestimate to the extent that En-positive neurons also die during these times.
From 50% onwards, pyknotic profiles had dense HRP-label, indicating
neuronal differentiation prior to death. Some profiles were En-negative at the
core, whereas others were En positive, confirming that neurons of both fates
are removed from the population (Fig.
5C,D). It was previously assumed that all pyknotic profiles late
in the lineage were cells of an interneuronal fate, in accordance with the
sequential lineage model (Thompson and
Siegler, 1993). In light of present data, this appears to be
incorrect. These events are further documented in a related study (M. V. S. S.
and X. X. J., unpublished). No evidence was found for cell death among GMCs or
GCs, as identified by a lack of HRP labeling, reinforcing the view that some
degree of neuronal differentiation precedes cell death.
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DISCUSSION |
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Our results are consistent with the first description of the four separate
progenitors (Bate and Grunewald,
1981). This contrasts with the view that the MP5 and MP6 progeny,
and probably also the MP4 progeny, are the earliest neurons produced by the
MNB (Condron and Zinn, 1994
).
It has also been reported that the MNB produces neurons from 28-33% and after
40%, and that during the intervening 7% gap, the MNB produces midline glia
(Condron and Zinn, 1994
;
Condron et al., 1994
). The 5%
gap in differentiation that we observe is roughly similar to this in timing
and extent, but we find a different cause: timing of neuronal differentiation
among progeny of the different precursors.
To understand how our own findings might be reconciled with earlier
reports, we ultimately focused on methodological issues. One point is readily
resolved. The slight difference in the timing of the `gap' in neuronal
production can be ascribed to differences in the antibodies used to label
neurons. The anti-HRP Ab detects new neurons at an earlier stage of
differentiation than does MAb 8B7, the latter having been used in prior
studies. A second point, that of the neuronal or glial identity of MNB
progeny, can also be resolved by considering methodology, but requires more
discussion. In an earlier study, midline lineages were assessed by injecting
the putative MNB with tracer at 28-29% neuromere development, and then
inspecting labeled progeny after a culture period of 72 hours
(Condron and Zinn, 1994). While
it might seem that such a direct approach would give a more accurate picture
than that afforded from the close study of labeled, fixed preparations, two
concerns are key. One is the identity of the injected progenitor. The other is
the timing of glial development.
The 28% time point is important because the MNB and MP6 are then adjacent, and of similar size. In fixed, antibody-labeled embryos, the two can be readily distinguished from each other by their intensities of En expression and by their positions relative to differentiating MP4 and MP5 progeny, but use of live embryos does not afford such markings. Thus, it is entirely plausible that in the prior lineage studies, the MNB was injected with tracer in some 28-29% embryos, whereas MP6 was injected in others. Indeed, these earlier data show two outcomes of injection. In one outcome, a single pair of neurons of unlike type contained the injected label. Both neurons bifurcated at the midline, but at separate anteroposterior positions, this being consistent with the morphological differences we see between the sibling MP6 progeny. Unintended injection of MP6, instead of the MNB, thus could have lead to the conclusion that these siblings arose early from the MNB, prior to a purported period of glial production. In the other outcome, a small group of GMCs or neuronal progeny contained the injected label, this consistent with an injection of the MNB and with the lineal development that we report.
Do the MNBs produce glia?
The timing of midline glial development is also important in assessing our
results relative to prior studies. We find that the MNB is surrounded by glia
by (at least) 28%, and the glial sheath is intimately associated with the MNB
by (at least) 34%. By an earlier view, the glia are produced from 33-40%, and
a glial sheath surrounds the MNB after 35%
(Condron and Zinn, 1994). In
support of these observations, injection of the MNB at 34% labels the midline
glial sheath only (or the sheath and neurons, depending on length of the
subsequent period of culture). We suggest the possibility that tracer entered
the already present glial sheath, which would necessarily be penetrated by an
injection microelectrode before it reached the MNB. This would label glial
cells directly, rather than via lineal conveyance from the MNB. Inadvertent
introduction of tracers into the glial surround, instead of or in addition to
injection of the MNB, might well be difficult to avoid, given their intimate
association.
Our data do not support the idea that En is involved in switching MNB
progeny from a neuronal fate to a glial fate. When En-antisense nucleotides
were injected into the MNB, the results were taken as evidence that, by
blocking En expression, the MNB was caused to produce extra neuronal progeny
at the expense of glial progeny (Condron et
al., 1994). A key methodological point is that neuronal somata
were counted by using 8B7-label. Such counts would underestimate the normal
population, however, because strong 8B7-label is transient in neuronal somata,
as discussed in the Results. Indeed, by using the more robust HRP-label we
counted about twice the number of neurons than were found by using the
8B7-label in normal embryos at the same stages
(Condron and Zinn, 1994
;
Condron et al., 1994
).
Moreover, our neuronal counts in normals were virtually identical to those
reported for the antisense-injected preparations. This suggests that neuronal
number was not altered by the antisense manipulation, rather that the number
of 8B7-labeled somata was increased. Midline neurons in the injected
preparations also had truncated neurites, and it is plausible that, absent
normal neurite elongation, the 8B7 epitope accumulated at the somata.
Normally, 8B7-label on somata wanes as neurites extend, as shown in the
present study. En-antisense nucleotides, introduced into the neuronal lineage
via injection of the MNB, might well disturb development of the En-positive
GMCs and neuronal progeny. A direct effect on En levels in the MNB is
unlikely, however, because (as we show) En expression in the MNB is negligible
by the time it is generating progeny. En-antisense injections also resulted in
a failure of normal glial development, this interpreted as the result of
switching the MNB fate to favor neuronal production. If En-antisense
nucleotides were inadvertently introduced into the already present glial
surround, this might well yield abnormal glial development as the glia
themselves are En-positive.
We find no evidence that the MNB produces glial progeny, but the
possibility cannot be formally excluded that at an initial division, a
precursor of the MNB gives rises to the MNB and to a glioblast. In
Drosophila, this pattern is seen in a limited number of NBs (or
neuroglioblasts) (Bossing et al.,
1996; Schmid et al.,
1999
; Schmidt et al.,
1997
). However, there is no evidence from Drosophila that
the MNB produces glia. Instead, the midline glia arise from separate glial
progenitors (Jacobs and Goodman,
1989
; Klämbt et al.,
1991
; Klämbt et al.,
1996
). Our evidence from grasshopper is consistent with this
scheme, reinforcing the view that grasshopper and Drosophila share
essential features of embryonic CNS development.
En expression and asymmetric fate in MP and MNB lineages
Progeny of the MPs4-6 are of asymmetric type. This pattern is again
consistent with what we know of midline development in Drosophila.
Three so-called VUM neurons in each embryonic segment are En-negative efferent
neurons, whereas three presumed siblings are En-positive, and of interneuronal
fate (Siegler and Jia,
1999).
The two types in grasshopper come to occupy respectively more dorsal, or
more ventral somata positions, trace separate midline bundles, and branch
bilaterally at separate anteroposterior locations. The three En-positive
interneurons pioneer the ventral region of the midline fiber tract, and
bifurcate at the PC. The three En-negative efferents pioneer the dorsal region
of the midline fiber tract, and grow anteriorly past the PC to bifurcate at
the AC. It was earlier reported that the two MP4 progeny were of like
morphology, and pioneered the dorsal region of the median fiber tract,
branching at the AC, whereas the two MP6 progeny (also of like morphology)
pioneered the ventral region of the median fiber tract, and branched at the PC
(Goodman et al., 1981), but
this appears not to be the case. Sibling progeny of the MNB lineage likewise
are of asymmetric type. This pattern of sibling pairs does not, however,
reflect the final composition of the MNB group. Substantial cell death
ultimately removes neurons of both types, though predominantly those of
En-negative fate. In abdominal neuromeres, the three surviving efferent
neurons (so-called DUM neurons) are almost certainly the three efferent
progeny of MPs4-6, and given this, it appears that all efferent progeny
generated by MNB abdominal lineages ultimately die. Pyknotic profiles were
surrounded by robust HRP-label, indicating that death occurred only after
neuronal differentiation had begun. The timing of these events suggests that
neuronal cell death occurs in some En-negative neurons before their axons exit
the CNS.
En expression levels change predictably during midline development. The
MPs4-6 have the highest level of En expression just before division. A high
level of En expression is maintained in one MP sibling, while in the other MP
sibling En is reduced to undetectable levels after 1-2% of development. The
MNB expresses En at its earliest stage, but never at the levels seen in the
MPs. En expression wanes to background levels by the time the MNB is producing
GMCs. The GMCs are En-negative as they arise from the MNB but gradually
develop En expression, which is strongest just before a particular GMC divides
to produce two GCs. En expression could regulate the expression of cell
adhesion molecules in the GMCs as it does in neurons
(Siegler and Jia, 1999),
perhaps to allow the most mature GMC to separate from the GMC column and
undergo division. Regulation of En expression in GMC sibling progeny is
similar to that in the MP4-6 progeny. Although MPs are sometimes referred to
as neuroblasts, our findings emphasize the similarity between MPs and GMCs, a
similarity that may be important in understanding the contrasting modes of
asymmetric division between neuroblasts, which act as stem cells, and other
CNS neuronal precursors, which undergo terminal divisions.
Differential En expression would be critical for the differentiation of
asymmetric neuronal types in the MP4-6 and MNB lineages. The gradual loss of
En in one member of a sibling pair coincides with the beginning of neuronal
differentiation, and is evident well before growth cone formation.
Differential En expression could then contribute to the different pathway
choices made by the neurons (Marie et al.,
2000). In Drosophila, En regulates the expression of
neuronal cell adhesion molecules, and misexpression of En in postmitotic
neurons grossly alters neuronal morphology
(Siegler and Jia, 1999
). As in
grasshopper, En expression occurs in interneurons, but not in efferent
neurons, further evidence for a role of En in the differentiation of neuronal
type (Siegler and Pankhaniya,
1997
; Siegler and Jia,
1999
; Siegler et al.,
2001
). The question remains unanswered as to the means by which
local or intersegmental interneuronal fate is determined. En expression
differs considerably among the mature interneurons, and may contribute to
pathway choice (Siegler and Pankhaniya,
1997
). A successive expression of several transcription factors
could confer a more specific ordering of phenotype
(Isshiki et al., 2001
).
A revision of midline lineages developmental and evolutionary
implications
The MNB is one of the longest-lived NBs in grasshopper and produces one of
the largest neuronal lineages. The MNB lineage was among the first of the NB
lineages to be studied in detail, and in sum the morphological,
electrophysiological and biochemical properties of its embryonic and adult
neurons are more thoroughly known than for other lineage groups in insects.
The MNB lineage of grasshopper has served as a basis for understanding how
neuronal types are produced broadly across lineages of the insect CNS
(Burrows, 1996;
Truman et al., 1993
).
The sequential model had seemed plausible, given that sibling neurons were
assumed to remain in proximity (Goodman
and Spitzer, 1979; Goodman et
al., 1980
). However, it is now clear that asymmetric neuronal
siblings from GMC and MP divisions are not positioned according to birth order
as previously thought, rather their somata come to occupy relatively dorsal or
ventral locations according to neuronal type. Their primary neurites are
wholly separated by neuronal type, tracing different midline tracts and
different commissures. Thus, neuronal morphology and position are not good
indicators of birth order within a lineage.
Our findings lead to a revised account of the MPs4-6 and MNB lineages
(Fig. 6). Evidence has long
been available from grasshopper, and then from Drosophila, that
sibling neurons of apparently unlike, or asymmetric type are produced from
MPs1-3 and from the first GMCs of some NB lineages
(Doe et al., 1988a;
Doe et al., 1988b
;
Goodman et al., 1981
;
Kuwada and Goodman, 1985
).
And, at least one postembryonic lineage in Manduca produces siblings
of two types (Witten and Truman,
1991
). These findings have not been incorporated into a more
general model of neuronal lineages, however; possibly for two reasons. In
contrast to other MPs, MPs4-6 were thought each to produce progeny of like
type (Goodman et al., 1981
).
And, in the few NB lineages with early progeny of apparently asymmetric type,
the range of neuronal types produced across the lineage is unknown. Without
this larger framework, it is hard to judge the significance of any phenotypic
difference. Differences between siblings might be ascribed to a particular
variation between neurons of the same basic type or they might, instead,
typify an essential asymmetry that is maintained across a lineage. The
neuronal progeny of embryonic NB lineages in Drosophila are now known
in some detail, and it is striking that two-thirds of lineages contain
interneurons as well as either motoneurons, or neurosecretory cells of an
efferent type (Schmid et al.,
1999
). Interneurons predominate in the extant lineages, but this
is not surprising in light of the extensive cell death we find among neurons
of efferent fate, and suspect the same to be true of Drosophila. Of
the remaining lineages in Drosophila, many generate interneurons
having two distinctly different axonal projections, either anterior or
posterior, or ipsilateral or contralateral. Both of these dichotomies could
represent an essential asymmetry in lineages that do not produce efferent
neurons; the MP2 siblings, for example, are asymmetric in that they have
either an anterior or a posterior interneuronal projection
(Spana et al., 1995
).
|
We suggest that the production of paired siblings of asymmetric neuronal
type is fundamental to insect neuronal lineages in the CNS. This view is
concordant with the asymmetry by which sense organs are generated in the
insect PNS. Furthermore, it parallels the essential role of asymmetry in the
unfolding of cellular and neuronal type in C. elegans, and in the
vertebrate CNS, as reviewed by Lu et al.
(Lu et al., 2000) and
references therein.
This idea has important implications for understanding and interpreting developmental and evolutionary events. During development, neuromeres across the CNS come to differ in size and composition, although each neuromere arises from a similar array of neuronal precursors. The means by which segmental specificity is achieved are largely unknown. In the MNB lineages, most interneurons survive, while only a minor faction of efferent neurons survive (this differing between thoracic and abdominal neuromeres). In effect, the lineages produce a large population of one neuronal type (interneurons), at the expense of overproducing another (efferent neurons).
Across insect evolution, the body appendages and their attendant sensory and motor apparati have been exquisitely formed and varied to match environmental needs. The first line of encounter, and thus the driving force for selection, is outward body form. The overproduction of central neurons, both of interneurons that integrate peripheral sensory input, and of efferent neurons that guide an outward motor response has a cost, but also a greater benefit. It confers considerable latitude to the CNS in matching outward demands, without necessitating fundamental changes in the lineal mechanisms by which different neuronal types are produced. Such a strategy offers simplicity and flexibility, providing a means by which neuronal populations in the insect CNS might be tailored to the wide diversity of peripheral structures across body segments, as well as tailored to the considerable diversity of body form and function that has emerged across the course of insect evolution.
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ACKNOWLEDGMENTS |
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