1 Department of Biology, University of Washington, Seattle, WA 98195, USA
2 School of Biological Sciences, University of Southampton, Southampton SO16
7PX, UK
* Author for correspondence (e-mail: jwt{at}u.washington.edu)
Accepted 14 July 2004
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SUMMARY |
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Key words: Neurogenesis, Metamorphosis, Neuronal architecture
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Introduction |
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In insects that have complete metamorphosis, like Drosophila and
the moth Manduca sexta, the NBs generate an initial set of neurons
that regulate larval behavior, but many then make a much larger set that have
an adult-specific function (Booker and
Truman, 1987; Truman and Bate,
1988
; Prokop and Technau,
1991
). These adult-specific neurons, most of which are born during
larval life, extend a primary neurite into the neuropil but then arrest. As
the larva grows, each NB accumulates a growing cluster of these arrested
immature neurons until the onset of metamorphosis when these cells show
intense sprouting as they find their adult synaptic targets. In this paper, we
have focused on the adult-specific lineages in the ventral CNS. The
fasciculated neurites arising from these lineages express the cell adhesion
protein, Neurotactin (de la Escalera et
al., 1990
), and they make a complex scaffold of neurite bundles
within the thoracic neuropils. Through the use of MARCM-based clones
(Lee and Luo, 1999
), we
identified the 24 lineages that make up the scaffold of a thoracic
hemineuromere. Unlike the early-born neurons that are strikingly diverse in
both form and function, the later-born cells in a given lineage are remarkably
similar and typically project to only one or two primary targets, which appear
to be the bundled neurites from other lineages. Correlated changes in these
patterns of projection and contact between segmental neuromeres suggest that
these initial contacts may denote future synaptic partners and functional
relationships amongst the lineages. This paper provides an overall view of the
initial connections that eventually lead to the complex connectivity of the
bulk of the thoracic neurons. It establishes a developmental framework from
which we will be able to understand the developmental rules that determine the
synaptic connectivity of the adult CNS.
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Materials and methods |
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Elav based clones: GAL4C155, hsFLP; FRT42B, tubP-GAL80/FRT42B, UAS-mCD8::GFP
Actin based clones: hsFLP; FRT42B, tubP-GAL80/FRT42B, UAS-mCD8::GFP; ActinGAL4
Tubulin-based clones: hsFLP, tubP-GAL80, FRT19A/FRT19A; UAS-mCD8::GFP, tubP-GAL4.
The actinGAL4 stock was a gift from B. Edgar, all other stocks were obtained from the Drosophila stock center (Bloomington, Indiana).
Generation of MARCM clones
Two heat shock regimes were used to generate MARCM clones. In the early
heat-shock regime, eggs were collected on grape juice plates for 2 hours, held
for 3 hours (both at 25°C) and then incubated for 1 hour at 37°C.
Hence, the embryos were heat shocked between 3 and 5 hours of embryogenesis.
In the late heat-shock regime, eggs were collected for 2 hours, held for 5
hours (both at 25°C), and then incubated for 1 hour at 37°C. Embryos
were therefore heat shocked between 5 and 7 hours of embryogenesis. Larvae
were reared on standard cornmeal food at either 25°C or room temperature
when precise staging was not a concern. Nervous systems were generally
dissected from larvae that were in the late 3rd instar or during
wandering.
Immunocytochemistry and in situ hybridization
Nervous systems were dissected from larvae and fixed in 3.7% buffered
formaldehyde for about 1 hour at room temperature and then washed three times
in PBS-TX [phosphate buffered saline (pH 7.8) with 1% Triton-X100]. Fixed
samples were blocked in 2% normal donkey serum (Jackson ImmunoResearch
Laboratories, West Grove, PA, USA) in PBS-TX for 30 minutes and then incubated
in various combinations of primary antibodies for 1 to 2 days at 4°C. In
preparations examining the relationship of the mCD8::GFP labeled clones to the
Neurotactin scaffold, the CNSs were incubated in a 1:50 dilution of an
anti-Neurotactin monoclonal antibody (F4A; a generous gift from Dr M. Piovant)
and 1:1000 dilution of a rabbit anti-mCD8 (Caltag Laboratories, Burlingame,
CA, USA) After washing out unbound primary antibodies, tissues were incubated
overnight at 4°C in a 1:500 dilution of FITC conjugated donkey anti-rabbit
IgG and Texas Red conjugated donkey anti-mouse IgG (Jackson ImmunoResearch
Laboratories, West Grove, PA, USA). After repeated washes with PBS-TX, tissues
were mounted on poly-lysine coated coverslips, dehydrated, cleared through
xylene and mounted in DPX (Fluka, Bachs, Switzerland).
For diaminobenzadine (DAB)-stained preparations the tissue was fixed as above. The tissue was incubated in 2 N HCl in PBS for 30 minutes and washed in PBS-TX three times. Fixed samples were blocked in normal 2% horse serum (Vector Laboratories, Peterborough, UK) for 1 hour and then incubated in a 1:250 dilution of anti GFP (Roche Diagnostics, Lewes, UK) overnight at 4°C After washing out unbound primary antibody, tissues were incubated overnight at 4°C in a 1:500 dilution of biotinylated horse anti-mouse IgG. After washing the tissue was incubated in a 1% solution of an avidin-biotin complex (Vector Laboratories, Peterborough, UK) for 2 hours. The tissues were washed and the antibody binding reveled by incubation in a 3% solution of diaminobenzidine and hydrogen peroxide.
Microscopy and image processing
Fluorescently stained nervous systems were imaged at 60x using a
BioRad MRC600 confocal microscope. z-stacks were collected with
optical sections at 1.5 µm intervals. In collecting the z-stacks,
the excitation wavelength was optimized for the respective fluorophore to
avoid bleed-through.
Raw data stacks were imported into NIH Image (http://rsb.info.nih.gov/nih-image/). Where necessary, adjustment to contrast and brightness were made to the entire data stack. Some nervous systems had single clones or widely spaced clones so that each could be easily viewed without interference. In many cases, though, there were multiple clones in a region and these obscured details in projected or rotated images. In these stacks, we would select a particular clone and use the lasso tool to remove the stained processes and cell bodies from other clones. This procedure would be carried multiple times on the same data stack, in each case isolating a different clone. Using Image J (http://rsb.info.nih.gov/ij/), we then made merges of the whole clonal array and of the individual clones with the same Neurotactin scaffold. This allowed us to determine the relationship of the various clones to one another and to the Neurotactin scaffold.
The data in the paper are typically presented as `thick-section' merges. We took 5-10 section portions of the Neurotactin stack and projected these as a two-dimensional image. The corresponding sections from the clone data stack were also projected as a flat image. The two projections were then combined in Photoshop (Adobe, San Jose, CA), with the Neurotactin image in red and the clone image overlaid in white.
Numbering of the lineages
We have been able to associate about half of the adult-specific lineages
with their embryonic NB. We decided not to use the embryonic designations
(e.g. 3-3 for the lineage from NB 3-3) for these lineages because we favored
having a consistent nomenclature at this time, rather than one that was mixed.
Generally the adult-specific lineages were numbered as they were identified
and we have not tried to relate the numbering to either position or projection
pattern.
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Results |
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In the thoracic and A1 neuromeres, the postembryonic lineages are situated from the ventral midline around to the dorsolateral boundary of the cellular rind (Fig. 1A). The relative insertion points of the neurite bundles into the neuropil are invariant and can be used to identify the individual lineages. Fig. 1I shows a thick section projection of the bundles emerging from the lineages in the ventral region of the rind. The relative position of these lineages is schematically depicted in subsequent figures along with the remainder of the dorsal and lateral lineages that are missing from the section in Fig. 1I. For some of lineages, we have also included data from the subesophageal ganglion.
Characteristics of the segmental lineages
The following description is based on 300 clones from individuals that
carried Elav-GAL4 based MARCM clones and were double stained for Neurotactin
(see Table S1 in the supplementary material). We analyzed an equivalent number
of Elav clones that were either fluorescently marked but without Neurotactin
labeling or were DAB-stained preparations. The variation in the number of
times that we identified a particular lineage probably reflects the timing of
when the neuroblast for the particular lineage started dividing in the embryo.
In a very few cases (e.g. lineage 7 in T1), we know that the lineage is
present despite our failure to recover clones in that segment, because we can
identify the Neurotactin bundle corresponding to that of lineage 7 in that
segment. A characteristic feature of each lineage is the pattern of projection
of the bundle(s) of neurites that emerge from the cluster of immature neurons.
We have given each of the 33 bundles at least a binary designation, including
a number (indicating its lineage of origin) and a letter to indicate whether
it projects ipsilateral (i) or contralateral (c). In the cases in which two
bundles terminate on the same side of the midline, we added a second letter to
denote dorsal (d), middle (m), ventral (v) or lateral (l) trajectories. Where
we use merged thick sections to illustrate the relationship of a clone to the
Neurotactin scaffold, the dorsal-most section is displayed at the top. We
omitted the binary designation in labeling the figures in cases in which a
lineage has only a single bundle. Additional information on each lineage is
available at
http://depts.washington.edu/nbatlas/.
Lineage 0
Lineage 0 (Fig. 2) is an
Engrailed positive lineage produced by the median unpaired neuroblast (NB 0).
A lineage 0 cell cluster is located at the ventral midline of segments S3
through A1. In T2, the neurites coming from the cluster form a single bundle
(0) that projects anterodorsally to the midpoint of the aI commissure where
the processes then splay out at about the level of the paired 2i bundles
(Fig. 2A,D). An identical
projection pattern is seen for the 0 bundle from the T3 and A1 clusters, but
in T1 the bundle does not project anteriorly and terminates at the level of
the pI commissure (Fig. 2A,C).
In S3, the 0 bundle also projects to the pI commissure but some fibers turn
anteriorly to form a more complex terminal projection
(Fig. 2B).
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Lineage 2
The cell cluster from lineage 2 is located near the midline on the anterior
margin of the neuromere (Fig.
1I). Fig. 4 shows
two lineage 2 clones that were hit in the same neuromeres. In Actin-based
clones, lineage 2 is associated with a cluster of larval neurons that lack
efferents and have a simple contralateral projection similar to that described
for the embryonic progeny of NB 2-1
(Schmid et al., 1999). Based
on the morphology of its larval siblings and its position in the neuromeres,
we have ascribed it to NB 2-1. Lineage 2 clusters are found only in segments
T1 to T3 and their projection pattern is identical in all segments. A single
neurite bundle (2i) projects dorsally from the cluster and then turns sharply
lateral when reaching the dorsal surface of the neuropil. Although the
processes do not cross the midline, they make up the majority of the aD
commissure.
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Lineage 3
Lineage 3 is an Engrailed-positive cluster situated on the posterior border
of the neuromere just lateral to cluster 12
(Fig. 1I) in S3 through A1.
Actin clones of lineage 3 include at least four motoneurons (the `U'
motoneurons) that project out the ipsilateral segmental nerve
(Fig. 5F), showing that this
cluster is produced by NB 7-1 (Landgraf et
al., 1997). In neuromere T3, a single neurite bundle (3i) projects
dorsally from the cluster but then splits into a dorsal (3id) and a lateral
(3il) bundle at the level of the pI commissure
(Fig. 5A-C). The 3id bundle
continues dorsally and terminates next to bundle 6cd as the latter bends
medially to form the pD commissure (Fig.
5A). The 3il bundle extends laterally and spreads anteriorly over
the dorsal region of the ventrolateral neuropil
(Fig. 5B).
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Lineage 4
The lineage 4 cell cluster is situated near the midline, just posterior to
cluster 10. It is present in segments T1 to T3 but it is not found in the
subesophageal or abdominal neuromeres. Actin-based clones containing the
larval siblings of lineage 4 include the motoneurons RP1,3,4,5 (data not
shown), which identify this lineage as being from NB 3-1
(Landgraf et al., 1997). This
adult-specific lineage produces a single neurite bundle
(Fig. 6) that projects
laterally along the ventral surface of the neuropil and terminates in the
ventrolateral neuropil posterior to the lateral cylinder
(Fig. 6B).
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The projection pattern of lineage 6 is the same in the all the thoracic neuromeres, although there is a reduction in the number of fibers in the 6cd bundle in T1. The A1 version of lineage 6 also makes a slightly smaller 6cd bundle, but its 6cm bundle is dramatically reduced (Fig. 8A,D,E) and was missing in some nervous systems. Two neurite bundles are also found in the S3 version of lineage 6, and in this case, the 6cm bundle is also greatly reduced relative to bundle 6cd (data not shown).
Lineage 7
Lineage 7 is a ventrolateral cluster in the anterior half of the
hemineuromere and is surrounded by clusters 13, 14 and 15. It is found in
segments T1 to A1 (Fig. 9). The
projection pattern of the neurons in lineage 7 is identical to the
interneurons that are produced by NB 3-3 during embryogenesis
(Schmid et al., 1999), and we
have ascribed this lineage to that NB. Fig.
9B-D shows an example of lineage 7 from A1 but the same projection
pattern is seen in the thoracic neuromeres
(Fig. 9A). The cluster produces
a single neurite bundle (7c) that extends across the midline as a bundle of
the aI commissure (Fig. 9C). In
thoracic neuromeres, the bundle from lineages 7 and 8 make up the portion of
the aI commissure located posterior to the ascending 2i bundles (e.g.
Fig. 1F,
Fig. 10B). Both lineage 2 and
lineage 8 are absent from A1 so the only components of the aI commissure that
remain are the bundles from lineages 7 and 18
(Fig. 9C). After crossing the
midline, bundle 7c extends anteriorly in a dorsal tract. We did not recover a
clone of lineage 7 in segment T1, but Neurotactin stained nervous systems show
that lineage 7 is present in T1 and also projects towards more anterior
segments. We could not tell with certainty whether lineage 7 is present in
S3.
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Lineage 9
This lineage is typically the most dorsal cluster in the anterior half of
the hemineuromere. The neurites from lineage 9 cluster to form a robust
ipsilateral (bundle 9i) and a sparse contralateral projection (9c)
(Fig. 11A). The neurites in
the 9i bundle extend ventrally to just below the intermediate commissure where
they then curve posteriorly to form the dorsomedial boundary of the lateral
cylinder (Fig. 1C,G;
Fig. 11E). The thin 9c bundle
projects down to the level of the ventral commissure where it enters the
commissure at an anterior level with the 1c bundle but then crosses over to
the more posterior commissural bundles (13c and 14c) prior to reaching the
midline (Fig. 11F). The bundle
then ends prior to reaching the lateral neuropil.
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Lineage 10
The cell cluster for lineage 10 is positioned just lateral to lineage 2
along the anterior border of the hemineuromere
(Fig. 1I). A single neurite
bundle (10c) extends dorsally from the cluster, forming the anterior ventral
arch, and then widens out towards the midline to form the floor of the aI
commissure (Fig. 12A-C). Most
neurites terminate soon after crossing the midline but a few extend laterally
and then turn either anteriorly or posteriorly through an intermediate level
of the neuropil. We obtained only one example of a lineage 10 clone, which was
in segment T2 (Fig. 12B). The
similarity of the Neurotactin projection for this lineage in all of the
thoracic segments, however, gives us confidence that this projection pattern
is also seen in segments T1 and T3. Lineage 10 is not found outside of the
thorax.
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Lineage 12
This Engrailed positive lineage is just lateral to the median lineage
(Fig. 1I) and is found in
S3-A1. Actin-GAL4 based MARCM clones show that this lineage has no motoneurons
associated with it and the larval interneurons neurons have projections
patterns that match the embryonic progeny of NB 6-1. The most complex
projection pattern is seen for the lineage 12 clusters in T1 and T2
(Fig. 14B-F). The neurite
bundle projects dorsally from the cluster and separates into contralateral
(12c) and ipsilateral (12i) bundles (Fig.
14F). Bundle 12c is the most posterior bundle of the posterior
ventral arch (which also contains bundles 6cm and 5c), and, after crossing the
midline in the pI commissure, the bundle dips slightly ventral and terminates
in a compact spray of arbor. As it projects dorsally, the 12i branch further
divides into middle (12im) and dorsal (12id) bundles at about the level of the
intermediate commissure (Fig.
14E). The 12im bundle runs along side the 11im bundle from lineage
11 and terminates along with this bundle at about the same level of the
intermediate neuropil (Fig.
14D). The 12id bundle continues to the dorsal-most region of the
neuropil where it ends along with the 3id and 11id bundles
(Fig. 14C).
In terms of its segmental morphology, lineage 12 is the most variable of all of the ventral lineages. We have examined 24 lineage 12 clones ranging from S3 to A1. In A1 (n=2), the neurons form a very thin 12c bundle and extend a 12i bundle that terminates in mid-neuropil, ventral to the normal bifurcation site. In T3 (n=6), half the lineages showed only the 12c bundle (Fig. 14A), whereas the other half also had a 12i bundle but one that terminated in intermediate neuropil as in A1. This lack of the dorsal projections of the 12i bundle is significant because T3 lacks lineage 11, which produces the bundles that the 12i sub-bundles contact in the intermediate and dorsal neuropils. In T2 (n=7), four of the clones showed the typical three bundles, but the remaining three had bundle 12c and 12id, which extended into its normal site in the dorsal neuropil but they lacked 12im. In T1, five out of six clones had the three bundles, with the remaining one showing a 12id, but not the 12im bundle. The lineage 12 cluster in S3 (n=3) completely lacks the contralateral 12c bundle and retains only bundle 12id.
Lineage 13
The cell cluster for this lineage is situated in the ventrolateral region
of the hemineuromere between lineages 7 and 5
(Fig. 1I). Larval neurons
associated with the adult-specific lineage include an ipsilaterally projecting
motoneuron and local interneurons with projections either ipsilaterally or
contralaterally through the anterior commissure (data not shown). These are
characteristic of either NB 3-4 or NB 4-4 [indistinguishable by Schmid et al.
(Schmid et al., 1999)]. The
adult-specific cluster produces two neurite bundles
(Fig. 15). The contralateral
projecting bundle (13c) contributes the most posterior bundle of the ventral
commissure. It projects to the ventrolateral neuropil and arborizes around the
posterolateral border of the lateral core
(Fig. 15B). The 13i bundle
projects dorsally and terminates in the dorsal region of the ventrolateral
neuropil, just lateral to the lateral cylinder
(Fig. 15C). Lineage 13 is
found only in the thoracic neuromeres and projection pattern is similar in
each.
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Lineage 19
The cell cluster for this lineage is situated dorsolaterally at the
posterior border of the hemineuromere. From its position and the expression of
Engrailed in the lineage we have attributed it to NB 7-4. The cluster gives
rise to two neurite bundles (Fig.
21B-E). The contralateral bundle (19c) extends across the midline
in the pI commissure and then bends dorsally to extend anteriorly in a dorsal
longitudinal tract (Fig.
21C,D). A few neurites can also be seen to extend anteriorly from
the bundle at other levels along the pI commissure. The ipsilateral bundle
(19i) rapidly splays out into a diffuse projection just lateral of the lateral
cylinder (Fig. 21E; bundles
8c, 8i and 7c ascend through the center of the lateral cylinder). Preparations
with double clones show that bundle 19i terminates in the near vicinity of the
fuzzy arbor from the lineage 15 motoneurons.
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Lineage 20
Lineage 20 is a ventrolateral lineage that includes two motor axons
(Fig. 22). Based on its
position, the presence of multiple efferents and similarity of larval cells to
those described by Schmid et al. (Schmid
et al., 1999), we have assigned the cluster to NB 5-4. Its neurite
bundle from the adult-specific cluster comes together with that from lineages
21 and 22 to make a short, dorsally projecting tract. A landmark associated
with this tract is the ascending 1i bundle from the next posterior segment
that curves around it (Fig.
22C). Immediately after passing bundle 1i, the 20i bundle bends
anterolaterally and the fibers splay out to fill in the ventral neuropil
posterolateral to the lateral cylinder
(Fig. 22B,C). Lineage 20 has a
similar projection pattern in all thoracic segments. It is missing from both
the subesophageal and abdominal neuromeres.
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Discussion |
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The MARCM clones analyzed in this study were induced early in
embryogenesis, and should include both the embryonic and postembryonic progeny
from a given neuroblast. This, indeed, was seen when Actin-GAL4 or tub-GAL4
was used as a driver to make the MARCM clones (e.g.
Fig. 5F). The diversity of
morphologies and strength of GFP expression in the larval neurons, however,
sometimes obscured some of the neurites arising from the associated
adult-specific cluster. When we generated similar clones using the purported
pan-neuronal driver line, elav [C155]
(Lin and Goodman, 1994), the
fully differentiated larval neurons in the clones typically failed to show GFP
expression but expression was strong in the arrested, adult-specific cells.
Although we do not know the reason that mature larval neurons fail to express
under these conditions, elav-based clones were invaluable for
determining the exact projection patterns of the clusters of adult-specific
neurons and how each contributed to the overall Neurotactin scaffold. Having
established the morphology of the adult-specific region of the lineage, we
could then return to MARCM clones generated using tub-GAL4 and Actin-GAL4
drivers to associate the neurons of adult-specific clusters with their larval
siblings. As the larval progeny of all of the embryonic neuroblasts have been
described (Bossing et al.,
1996b
; Schmidt et al.,
1997
; Schmid et al.,
1999
), the larval neurons aided us in identifying the embryonic
neuroblast responsible for many of the adult-specific clusters.
The early neurons generated by a given NB typically show a great diversity
in terms of their type and their axonal projections (e.g. Ishiki et al., 2001;
Pearson and Doe, 2003).
Indeed, the projection patterns of the daughter cells can change dramatically
from one GMC to the next [e.g. for the early neurons of the median neuroblast
lineage, see Goodman and Spitzer (Goodman
and Spitzer, 1979
)]. Later born cells, though, appear to be much
more similar in their morphologies, transmitters and functions
(Shepherd and Laurent, 1992
;
Witten and Truman, 1991
;
Burrows and Siegler, 1982
).
The present study shows that the similarity in late-born progeny is a general
rule for all lineages. Although each NB may show a high degree of diversity in
the first few neurons that it produces, the vast majority of their progeny are
similar in their pathfinding decisions, with typically only one or two initial
targets for the neurites that leave a cluster. Indeed, we find only 33 major
projection patterns for the thousands of neurons that are born within a
thoracic hemineuromere.
The diversity of phenotypes in the early born cells of a lineage is
accomplished through the sequential expression of a series of transcription
factors (hunchback, kruppel, pdm and castor) that are passed
from the NB to successive GMCs (Kambadur
et al., 1998; Brody and
Odenwald, 2000
; Isshiki et
al., 2001
). This molecular specification of unique identities
imposed by the neuroblast on the first few neurons in a lineage appears to be
lacking in the later born neurons, all of which express grainyhead
(Brody and Odenwald, 2000
). We
suspect that the transition from uniquely specified GMCs to ones that express
the same transcription factor marks the transition from generating unique
individual neurons to generating neuronal classes. For the latter cells,
interaction with other neurons, rather than factors supplied by their NB, may
then be essential for establishing identity within their neuronal class. It
should be noted that the transition between uniquely identified neurons to
neuronal classes does not necessarily lie at the dividing line between the
embryonic and postembryonic phases of proliferation. By feeding larvae on diet
containing bromodeoxyuridine (BUdR) from the time of hatching, we have labeled
all of the neurons that are born during larval growth. Analysis of Elav-based
MARCM clones in these larvae showed some lineages in which some of the
developmentally arrested neurons were unlabeled and, hence, were born prior to
hatching. These were always the neurons in the clone that were nearest the
neuropil (i.e., the oldest cells) (J.W.T. and D.W.W., unpublished). Hence, the
NBs do not necessarily stop dividing after they make the neurons that will be
used in the larva, and they may depend on an extrinsic signal to terminate
their embryonic phase of neurogenesis. These embryonically born cells may
serve as pioneers to guide the growth of postembryonic members of their
lineage.
An interesting feature of the adult-specific neurons is that each extends
an initial neurite to a lineage-specific location but then their development
stalls until pupariation. As illustrated in the developing hippocampus
(Bagri et al., 2003), a
developing neuron often sends out a single, unbranched process with a growth
cone to navigate to an initial target, followed by interstitial sprouting then
enables interactions with secondary targets. Contact with the initial target
may persist or it may be lost through stereotyped pruning but connections with
final targets are often then refined through local cell-cell interactions. In
the adult-specific neurons in Drosophila, the period of developmental
arrest separates axon pathfinding and contact with the initial target from the
phase of interstitial sprouting to secondary targets. This arrest is
terminated at the start of metamorphosis, when the neurons show a profuse
sprouting, accompanied by the appearance of the broad-Z3
transcription factor (B. Zhou, D.W.W., J.W.T. and L. M. Riddiford,
unpublished), and the onset of nitric oxide (NO) sensitivity (S. Gibbs, D.
Currie and J.W.T., unpublished). The latter observation is especially
interesting because studies on other insect neurons show that the onset of NO
sensitivity occurs as a neuron shifts from pathfinding to interacting with its
synaptic targets (Truman et al.,
1996
; Ball and Truman,
1998
; Gibbs and Truman,
1998
). The appearance of NO sensitivity at the termination of
arrest suggests that the neurons have switched into a new developmental mode
in which interactions with future synaptic partners become of prime
importance.
Hence, the larval CNS just prior to metamorphosis gives us an unprecedented snap-shot of neuronal development. Thousands of neurons are arrested at their initial targets awaiting the hormonal signals that will initiate secondary sprouting. This probably represents a watershed in the development of the CNS. Up to this point in development, the identity of the neurons and their growth decisions may have been relatively `hard-wired' by genetic information supplied by the NB and the ganglion mother cell. After this point, interactions with their primary and secondary targets probably dominate in shaping the final phenotypes of the cells.
Segmental variation and its functional implications
The map of initial contacts depicted in
Fig. 26C is undoubtedly not a
complete description of all of these contacts. In addition, at this time we
cannot know the polarity of the contacts, i.e. who will be presynaptic and who
will be postsynaptic. Nevertheless, this map probably provides a broad
overview of the first step in establishing the connectivity for the bulk of
the thoracic neurons. These initial contacts acquire some functional
importance when we consider the segmental variation in their pattern
(Fig. 27). The patterns in
neuromeres T1, T3 and A1 are compared with the situation in T2, as this is the
only segment that possesses the full complement of 24 postembryonic lineages.
Importantly, many of the segmental changes involve coordinated changes in the
lineages that project to the same region of the neuropil. The most obvious
example involves the lineages associated with the ventrolateral neuropil.
These include the motor lineage (lineage 15) that makes exclusively
motoneurons and projects to a leg imaginal disc. Lineage 15 is confined to the
thoracic neuromeres as are nine other lineages that send their neurite bundles
exclusively to the ventrolateral neuropil. With one exception, these lineages
show no obvious variation in their projection patterns between the three
thoracic neuromeres. The only lineage that shows a variable projection pattern
is lineage 1, which also has initial targets in two adjacent neuromeres.
Accordingly this lineage retains its homosegmental projection (bundle 1c) in
T1 but it lacks the 1i bundle (i.e. no bundle projects to the SEG). All of the
lineages that project to the ventrolateral neuropil are absent from A1, with
again the exception of lineage 1. The lineage 1 neurons arising in A1, though,
all project to the T3 neuropil (via bundle 1i) and the homosegmental 1c bundle
is missing. Our identification of the lineages in the subesophageal neuromeres
is not complete but it appears that most, if not all, of these lineages are
also lacking from the SEG. Apart from lineages that project exclusively to the
ventrolateral neuropil, there are a few lineages, like lineages 3 and 19 that
have one bundle projecting to this neuropil and another projecting into more
dorsal regions. This is especially interesting in the case of lineage 19
because its 19i bundle makes contact with the expanded area of the lineage 15
bundle and therefore may represent premotor interneurons. These ventrolateral
projections, though, are missing in the A1 version of lineages 3 and 19 (see
Fig. 5C for lineage 3). The
uniformity of projection patterns within the thorax and their absence outside
of this region of the body suggests that all of the lineages that project to
ventrolateral neuropil make neurons involved with the sensory or motor
requirements of the legs. This functional interpretation is supported by the
fact that lineage 14 is one of the above lineages and its proposed homologues
in grasshoppers (from NB 4-1) process input from leg mechanosensory hairs
(Shepherd and Laurent, 1992)
and integrate locomotor reflexes of the leg (reviewed by
Burrows and Newland,
1997
).
Although the ventrolateral projections are relatively stable within the thoracic neuromeres, projections to intermediate and dorsal neuropils show striking segmental variation. For example, lineage 11 is absent from T3 and the two lineages that send neurite bundles that terminate next to those of lineage 11 in more anterior segments, have these bundles reduced (bundle 3id in Fig. 5) or missing altogether (the 12im and 12id bundles of lineage 12, Fig. 14) in this segment (Fig. 27). T1 also has its unique set of changes. In T2 and posterior, the 0 bundle from the median NB projects to the aI commissure and appears to terminate between bundle 10c (ventral to it) and bundle 18c (dorsal). The 18c bundle is missing in T1 and we see that bundle 0 is redirected to the pI commissure (Fig. 2). T1 also shows a marked reduction in the number of bundles that project to anterior neuromeres; bundle 18c is missing and bundle 19c is greatly reduced to only a few fibers. Thus, the neurons in the 18c and 19c bundles may be involved in coordination within the thorax rather than taking information to higher centers in the head. We do not find obvious glial structures at the sites where the neurite bundles terminate. The correlated loss of converging bundles (such as seen for 12id, 3id and 11id in T3), suggest that the initial targets for the neurites in a bundle from one lineage may be bundles from other lineages. The map in Fig. 26C is the first attempt to identify the lineage-level rules that are used for establishing the initial connectivity map in the thoracic CNS. Whether these initial contacts are maintained and how they relate to secondary targets remains to be determined.
Our preliminary observations of embryonic induced single and double cell
clones in lineage 6 show that in single neuron clones there is a single
neurite that is either in the 6cm or 6cd bundle. By contrast, two neuron
clones (arising from a GMC) show a neurite in both bundles. This suggests that
the two bundles are built up by each GMC producing two daughters, one that
chooses one pathway and one that chooses the other. While it obviously needs
to be tested, we expect that this pattern will hold for all of the lineages
that have bundles projecting to two initial targets. Interestingly, in the
cases in which one bundle is lost in a given segment (1i in T1, 12id and im,
and 3id in T3; and 19i and 1c in A1) the cell cluster in that segment is
markedly smaller that in other segments. A possible mechanism to explain the
segmental difference is that cell death shapes the projection pattern by
having the inappropriate daughter cell die after its birth. Studies of the
median lineage in grasshopper embryos show the importance of divergent sibling
fates and cell death in shaping features of that lineage
(Thompson and Siegler, 1993;
Jia and Siegler, 2002
).
Conclusions
The results from this study have developmental, behavioral and evolutionary
implications. Previous studies on the ventral ganglia (e.g.
Broadus and Doe, 1995) and the
brain (Urbach and Technau,
2003
) show that the neuroblasts express a striking diversity of
transcription factors and signaling molecules. Some of these molecules are
involved in the establishment of the unique identity of the neuroblasts (e.g.
Bhat, 1996
) and their
early-born progeny (Isshiki et al.,
2001
). Others, though, may function later in directing patterns of
connectivity (e.g. Bossing et al.,
1996a
). It has been difficult to determine the latter, however,
because projection patterns and potential targets were unknown for the vast
majority of neurons in the lineage. Our study indicates that the first step in
establishing the extreme complexity of CNS connections involves a rather
simple set of rules, with the bulk of the neurons of a given lineage following
one or two projection paths. At this time, we do not know if the 33 different
projection trajectories that we see in the thoracic neuropil are the product
of just 33 individual neurons per hemineuromere that pioneer the track for the
rest of their lineage or if all of the adult-specific neurons follow the same
set of cues to their initial targets. Irrespective of how they navigate their
path, the initial connectivity patterns
(Fig. 26C) suggest that
neurons in one lineage use other lineages as their targets. This information
should help us understand the roles of patterning genes such as
wingless and hedgehog in establishing connectivity and
neuronal properties within the CNS.
The elegant studies of the neural circuitry underlying sensory to motor
coordination in the legs of grasshoppers (reviewed by
Burrows and Newland, 1997)
showed that functionally related neurons were clustered, and some, indeed are
siblings that come from the same neuroblast
(Shepherd and Laurent, 1992
).
The uniformity of initial projections that we see within each of the
adult-specific lineages leads us to speculate that each neuroblast is devoted
to making a very small number of functional neuronal types, with the noted
exceptions of the early-born cells that have unique identities sculpted by the
expression of hunchback, kruppel, etc. Changes in specific behavioral
functions between species might then be reflected in selective alterations in
the particular lineages whose neurons participated in that behavior. One
possible illustration of this is in the shift from primitively wingless
insects to those that can fly was accompanied with marked increase in neuronal
progeny in only 14 out of the 31 thoracic lineages
(Truman and Ball, 1998
).
Indeed, the later born neurons in some subsets of lineages may co-evolve
because these cells are functionally connected. Although there has been only
minor differences in the neuroblast arrays when one compares grasshoppers to
Drosophila (Broadus and Doe,
1995
), some of the neuroblasts have changed the blend of
transcription factors that they express. It will be interesting to determine
if these changes do indeed reflect a change in identity of the neuroblast or
whether it reflects an alteration in instructions as to how these neurons
should connect.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/20/5167/DC1
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