First published online September 30, 2004
doi: 10.1242/10.1242/dev.01371
Development 131, 5167-5184 (2004)
Published by The Company of Biologists 2004
Developmental architecture of adult-specific lineages in the ventral CNS of Drosophila
James W. Truman1,*,
Hansjürgen Schuppe2,
David Shepherd2 and
Darren W. Williams1
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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In Drosophila most thoracic neuroblasts have two neurogenic
periods: an initial brief period during embryogenesis and a second prolonged
phase during larval growth. This study focuses on the adult-specific neurons
that are born primarily during the second phase of neurogenesis. The
fasciculated neurites arising from each cluster of adult-specific neurons
express the cell-adhesion protein Neurotactin and they make a complex scaffold
of neurite bundles within the thoracic neuropils. Using MARCM clones, 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 adult specific cells in a given lineage are
remarkably similar and typically project to only one or two initial targets,
which appear to be the bundled neurites from other lineages. Correlated
changes in the contacts between the lineages in different segments suggest
that these initial contacts have functional significance in terms of future
synaptic partners. This paper provides an overall view of the initial
connections that eventually lead to the complex connectivity of the bulk of
the thoracic neurons.
Key words: Neurogenesis, Metamorphosis, Neuronal architecture
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Introduction
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Developing nervous systems face the challenge of generating a wide
diversity of neuronal types and then establishing precise patterns of synaptic
connectivity between these cells. Neuronal precursor cells in the CNS have a
major influence on the properties of the neurons that they produce, but this
influence can change through time. For example, in the vertebrate cerebral
cortex (Desai and McConnell,
2000
) and retina (Cepko et
al., 1996) neuronal precursor cells initially have the competence
to produce a variety of neuronal types but this competence becomes
progressively reduced as proliferation progresses. This dependence of neuronal
phenotypes on the properties of their precursor cell reaches its extreme in
the developing CNS of insects. In these animals, the precursor cells [called
neuroblasts (NBs)] are organized in a stereotyped array of rows and columns,
with each stem cell having a unique identity as determined by position
(Bate, 1976;
Doe and Goodman, 1985) and
gene expression (Doe, 1992).
The neurons arising from each NB are highly diverse but unique to that stem
cell (Bossing et al., 1996b;
Schmidt et al., 1997;
Schmid et al., 1999). The
phenotypic diversity in the early progeny of each NB is specified by the
sequential expression of the transcription factors Hunchback, Kruppel, PDM and
Castor (Kambadur et al., 1998;
Brody and Odenwald, 2000;
Isshiki et al., 2001), but
fairly early in their lineages, the NBs lose the competence to respond to
these factors (e.g. Pearson and Doe,
2003) and the diversity of cell types that they can produce
becomes progressively restricted. Although the early diversity of neurons
within these lineages has been extensively studied, we know less about the
phenotypes of later cells. At present, the entire set of neurons for only one
lineage, the median lineage in grasshoppers, is known. After a highly diverse
initial set of progeny, the later born cells are quite similar, being divided
into two large classes of interneurons
(Thompson and Siegler, 1991),
an observation entirely consistent with the idea of restricted phenotypic
diversity later in a lineage. The key issue here is whether this example is
the rule or the exception.
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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Fly stocks
The MARCM technique was used, in which the FLP/FRT system induced clones
that lacked GAL80, a suppressor of GAL4, to make CD8::GFP-labeled
clones in an unlabeled background (Lee and
Luo, 1999). The three GAL4 drivers used during the course of this
work were Elav, Actin and Tubulin. The following genotypes were generated.
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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Neurotactin scaffold in the ventral CNS of larval Drosophila
The identification of the multicellular clones was facilitated by
referencing each clone to Neurotactin-positive bundles in the ventral CNS.
Neurotactin is a heterophilic cell adhesion protein and the fasciculated
neurites of the immature neurons express it highly on their surface
(Barthalay et al., 1990;
de la Escalera et al., 1990).
The Neurotactin staining revealed a complex scaffold of neurite bundles that
included all of the arrested postembryonic neurons in the CNS. Nervous systems
were double labeled so that we could identify which MARCM clones were
responsible for every component of the scaffold. The identity of the neurite
bundles and the landmarks that are especially useful for their identification
are summarized in Fig. 1. In
cross-section, Neurotactin stained bundles cross the midline in ventral,
intermediate and dorsal commissures (Fig.
1A). The intermediate commissure
(Fig. 1F,G contains the most
bundles and is divided into anterior (aI) and posterior (pI) sections. The
dorsal commissure (Fig. 1E) has
a thick posterior component (pD) and a poorly developed anterior section (aD).
Ventrally, there is only an anterior (aV) commissure
(Fig. 1H). Another prominent
neuropil landmark that we used is the `ventral arch' (VA,
Fig. 1A). It extends from
selected ventral lineages up to the intermediate commissure. Three bundles
make up the posterior ventral arch, whereas a single bundle forms the anterior
ventral arch.
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Fig. 1. Features of the Neurotactin-positive bundles in the thoracic CNS of larval
Drosophila. (A) A thick transverse projection of the bundles in one
thoracic neuromere. Brackets show the levels of the dorsal (D), intermediate
(I) and ventral (V) commissures; VA, ventral arch; tinted region highlights
the right ventrolateral (VL) neuropil. (B) Schematic representation of a cross
section through the neuropil, showing positions of the commissures,
ventrolateral neuropil and dorsal longitudinal tracts (DT). (C,D) Dorsal views
of confocal sections through the VL neuropils in T2 and T3 of a nervous system
immunostained for (left) Neurotactin (magenta) and with phalloidin (green).
Phalloidin staining shows the fine fibrous neuropil characteristic of the VL
neuropil, which contains a poorly stained core, the lateral cylinder (LC),
that is bounded by the Neurotactin bundles 9i and 1c (right). Several bundles
ascend through the cylinder. (E-I) Paired images of thick stack projections of
the T2 and T3 neuropils from the levels indicated on (A). The lineage bundles
are labeled on the negative version (right panel); the next ventral section is
also included as a `ghost' to aid in alignment of the bundles. Brackets show
the anterior (aD) and posterior (pD) dorsal commissures, the anterior (aI) and
posterior (pI) intermediate commissures, and the anterior ventral (aV)
commissure.
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Over half of the lineages project their neurite bundles to an area of fine
fibrous neuropil in the ventrolateral region of each thoracic neuromeres.
Phalloidin staining shows a dense packing of fine processes in this neuropil
(Fig. 1C,D). A prominent
feature within this neuropil is a structure that we called the `lateral
cylinder', which lacks fine processes. Neurotactin staining shows that the
lateral cylinder is bounded on its dorsomedial side by bundle 9i and on its
ventrolateral side by bundle 1c. It also contains 5 neurite bundles (7c, 8c,
8i, 15i, and 16i) that project dorsally through its core.
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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Fig. 2. Characteristics of median neuroblast lineage (lineage 0). (A) A thick
section projection of confocal slices showing the Neurotactin scaffold between
the ventral and intermediate commissures. The neurite bundle from lineage 0
projects to the level of the aI commissure up through T2, but is redirected to
the pI commissure in T1. Posterior ventral arch (pVA) is labeled in T3 but is
also present in T2 and T1. `0' identifies the ventral base of bundle 0 in each
segment. (B-D) Ventral views of projections of full lineage 0 clones in the
(B) S3, (C) T1 and (D) T3 neuromeres. Diagrams show the trajectory of the
neurite bundle in the Neurotactin scaffold, and the position of the lineage 0
cluster relative to the other clusters in the hemisegmental array. Commissure
abbreviations as in Fig. 1
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Lineage 1
The cell cluster for lineage 1 (Fig.
3) is located ventrolaterally along the anterior boarder of the
T1-A1 neuromeres. Lineage 1 is Engrailed positive, which identifies it as
belonging to NB 1-2 (Broadus and Doe,
1995). Bundle 1c also has the projection pattern seen for some of
the local interneurons generated by this NB
(Schmid et al., 1999). In T3,
the cell cluster produces two neurite bundles
(Fig. 3A). The contralateral
bundle (1c) loops dorsally over the bundle 17i and projects across the midline
as the most anterior bundle in the aV commissure. It then curves around the
anteriolateral border of the lateral cylinder in the ventrolateral neuropil.
Lineage 1 is the only lineage that has a major projection to the ventral
neuropil in an adjacent segment. The ipsilateral bundle (1i) projects
anteriorly to the next ventrolateral neuropil, where it splays out in a
ventral domain posterolateral to the lateral cylinder. Along its anterior
path, bundle 1i curves around the ascending neurite bundles from lineages 20,
21 and 22.
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Fig. 3. Characteristics of lineage 1 (NB1-2). (A) Dorsal view of the projection of
a lineage 1 clone in T3. (B,C) Thick section projections showing the
relationship of the neurite bundles from lineage 1 to features of the
Neurotactin scaffold (red) in the ventral neuropil. The contralateral bundle
(1c) projects to the contralateral ventrolateral neuropil, whereas the
ipsilateral bundle (1i) projects to the ventrolateral neuropil in T2.
Progressively ventral sections. (D) Lineage 1 in T1 lacks the anterior 1i
bundle. (E) Lineage 1 in A1 lacks the 1c bundle. Numbers identify neurite
bundles from other lineages. Diagrams as in
Fig. 2. Commissure
abbreviations as in Fig. 1.
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In T2, lineage 1 has an anatomy that is identical to that seen in T3. In
T1, however, the lineage has bundle 1c but the ipsilateral bundle (1i) is
missing (Fig. 3D). By contrast,
in A1, the ipsilateral bundle is present, while the contralateral bundle is
absent (Fig. 3E). Lineage 1,
therefore, appears to be comprised almost exclusively of neurons projecting to
thoracic sensory neuropils, most likely related to the legs.
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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Fig. 4. Characteristics of lineage 2 (NB 2-1). (A) Ventral view of the projection
of paired lineage 2 clones in the T2. (B-D) Thick section projections showing
the relationship of the clone to (B) dorsal, (C) intermediate and (D) ventral
features of the Neurotactin scaffold (red). Numbers identify neurite bundles
from other lineages. Diagrams as in Fig.
2. Commissure abbreviations as in
Fig. 1.
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The 2i bundles are important landmarks in the Neurotactin scaffold. Within
the aV commissure, the dorsally projecting 2i bundles divide the commissural
bundles with the bundles from lineage 1 being anterior and those from lineages
13 and 14 being posterior (Fig.
1H, Fig. 4D). For
the aI commissure, the lineage18 bundles crosses anterior to the 2i bundles,
whereas lineages 7 and 8 cross behind them
(Fig. 1F,
Fig. 4C).
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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Fig. 5. Characteristics of lineage 3 (NB 7-1). (A,B) Thick section projections
showing the relationship of the neurite bundles of lineage 3 clone from C to
(A) dorsal and (B) ventral features of the Neurotactin scaffold (red). (C)
Ventral view of the projection of a lineage 3 clones in the T3. (D) The
intermediate to dorsal region of lineage 3 in T2, showing the expanded 3id
bundle. (E) A1 version of lineage 3 lacking the 3il bundle. (F) An Actin-GAL4
based MARCM clone of lineage 3 showing larval neurons as well as the
adult-specific cells. The neurons with bright cell bodies are four of the `U'
motoneurons made by NB 7-1. Numbers identify neurite bundles from other
lineages. Diagrams as in Fig.
2. Commissure abbreviations as in
Fig. 1.
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In A1, lineage 3 lacks the 3il bundle and only bundle 3id is present
(Fig. 5E) and it ends next to
bundle 6cd as in T3. In segments T1 and T2, the termination of bundle 3id is
more diffuse than seen in the posterior neuromeres
(Fig. 5D). This altered
terminal projection is associated with the presence of bundles 11id and 12id,
which are confined to the T1 and T2 neuromeres.
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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Fig. 6. Characteristics of lineage 4 (NB 3-1). (A) Ventral view of the projection
of a lineage 4 clone. (B-D) Thick section projections showing the relationship
of the neurite bundle from lineage 4 to features of the Neurotactin scaffold
(red) in the ventral neuropil. (B-D) Progressively ventral slices. Numbers
identify neurite bundles from other lineages. Diagrams as in
Fig. 2. Commissure
abbreviations as in Fig. 1.
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Lineage 5
Lineage 5 has a neuronal cluster situated in the ventrolateral region of
the neuromere and is found in segments S3 to T3. The cluster produces a single
neurite bundle (5c) that projects medially along the ventral neuropil and then
bends dorsally as the anterior-most bundle of the posterior ventral arch
(along with 6cm and 12c; Fig.
7B,C). The bundle broadens into a flattened projection that covers
most of the anterior half of the pI commissure. Small neurites extend
anteriorly on either side of the commissure
(Fig. 8A,B). The lineage
located in S3 has a more pronounced anterior projection when compared with its
thoracic counterparts (not shown).
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Fig. 7. Characteristics of lineage 5. (A) Ventral view of the projection of a
lineage 5 MARCM clone in T1. (B-D) Thick section projections showing the
relationship of the neurite bundle from lineage 5 to features of the
Neurotactin scaffold (red) in intermediate and ventral neuropils.
Progressively ventral slices. Numbers identify neurite bundles from other
lineages. Diagrams as in Fig.
2. Commissure abbreviations as in
Fig. 1.
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Fig. 8. Characteristics of lineage 6 (NB 5-2). (A) Ventral view of the projection
of lineage 6 MARCM clones in T3 and A1. (B-E) Thick section projections
showing the relationship of the neurite bundles from lineage 6 to features of
the Neurotactin scaffold (red). The relationship of the bundles to structures
in the (B) dorsal, (C,D) intermediate and (E) ventral neuropils. In the
thorax, bundle 6cm forms the middle bundle of the posterior ventral arch and
then projects anteriorly in a dorsal tract; bundle 6cd forms the pD
commissure. Numbers identify neurite bundles from other lineages. Diagrams as
in Fig. 2. Commissure
abbreviations as in Fig. 1.
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Lineage 6
Lineage 6 is a medial lineage situated just anterior to lineage 12
(Fig. 1I) and is found in
segments S3 to A1. It is Engrailed negative and, hence, is from a NB row
anterior to row 6. Based on its medial position and the large number of cells
that it contributes to the posterior commissure (see
Schmid et al., 1999), we have
assigned this lineage to NB 5-2. In the thorax, the lineage 6 cluster produces
two neurite bundles, 6cm and 6cd, both of which project to contralateral
neuropil (Fig. 8A). Bundle 6cm
is the middle bundle of the posterior ventral arch
(Fig. 8D,E); it crosses the
midline in the pI commissure and then projects anteriorly in a dorsal
longitudinal tract (Fig. 8C).
Bundle 6cd extends more dorsally and then crosses the midline as the major
bundle of the pD commissure (Fig.
8B). The neurites extend to the lateral border of the pD
commissure and then dip ventrally before they terminate at the level of the
6ci bundle (Fig. 8B,C).
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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Fig. 9. Characteristics of lineage 7 (NB 3-3). (A) Ventral view of the projection
of lineage 7 MARCM clones from the T2 and T3 neuromeres. (B) Lineage 7 clone
from A1. (C,D) Thick section projections showing the relationship of the
neurite bundle from lineage 7 to features of the Neurotactin scaffold (red) in
the (C) intermediate and (D) ventral neuropils. Numbers identify neurite
bundles from other lineages. Diagrams as in
Fig. 2. Commissure
abbreviations as in Fig. 1.
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Fig. 10. Characteristics of lineage 8. (A) Ventral view of the projection of a
lineage 8 clone. (B,C) Thick section projections showing the relationship of
the neurite bundles from lineage 8 to features of the Neurotactin scaffold
(red) in (B) intermediate and (C) ventral neuropils. Bundle 8c is one of the 3
paired bundles that constitute the aI commissure; bundle 8i projects to the
most dorsal regions of the ventrolateral neuropil. Numbers identify neurite
bundles from other lineages. Diagrams as in
Fig. 2. Commissure
abbreviations as in Fig. 1.
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Lineage 8
Lineage 8 is a ventrolateral cluster situated just anterior to the lineage
15 cluster in the anterior half of the hemineuromere
(Fig. 1I). It is present only
in segments T1 through T3. The neurite bundle leaving the cell cluster
immediately splits into two bundles (8c and 8i) both of which extend dorsally
through the lateral cylinder (Fig.
10A-C). Bundle 8c exits the cylinder dorsal to bundle 9i, extends
across the midline as part of the aI tract just posterior to the ascending 2i
bundles (Fig. 10B), and
terminates shortly after crossing the midline. The ipsilateral bundle 8i
extends dorsally to about the level of the intermediate commissure and then
bends lateroventrally into the dorsal region of the ventrolateral neuropil,
just anterior to the lateral cylinder
(Fig. 10C). The bundle also
contains a single peripheral axon that continues out of the CNS. The
projections are similar in all three thoracic neuromeres.
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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Fig. 11. Characteristics of lineage 9. (A) Ventral view of the projection of MARCM
clones of lineage 9 in the T2 and T3 neuromeres. (B) Lineage 9 clone from A1.
(C) A thick section projection of confocal slices showing the ventral
commissures in T3-A3. In T3, the bundle is the thinnest of the four paired
bundles that constitute the aV commissure; it is the only aV bundle that is
found in the abdominal segments. (D-F) Thick section projections showing the
relationship of the neurite bundles from lineage 9 to features of the
Neurotactin scaffold (red) in intermediate (D,E) and ventral (F) neuropils.
Diagrams as in Fig. 2.
Commissure abbreviations as in Fig.
1.
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In T1 lineage 9 has an additional, thin contralateral bundle that extends
across the aI commissure anterior to the lineage 2 bundles (not shown). This
second contralateral projection was seen in 4 of 6 T1 lineage 9 clones but was
absent from the 11 clones recovered from segments T2 and T3. We have not
managed to identify this lineage in the subesophageal neuromeres. The size of
lineage 9 is greatly reduced in A1 (Fig.
11B) with a very small 9i bundle. The 9c bundle is also reduced to
only a few neurites and is the last vestige of the ventral commissure bundles
persisting in the abdomen. Lineage 9 is also found in the abdominal neuromeres
posterior to A1 (Fig. 11C).
There are three adult-specific lineages in the abdominal neuromeres posterior
to A1 (Truman and Bate, 1988).
Lineage 9 is the largest one and the only one of the three that we could
identify with certainty.
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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Fig. 12. Characteristics of lineage 10. (A) Ventral view of the projection of a
lineage 10 clone. (B,C) Thick section projections showing the relationship of
the neurite bundle from lineage 10 to features of the Neurotactin scaffold
(red) in (B) intermediate and (C) ventral neuropils. Bundle 10c is the only
bundle of the anterior ventral arch. Numbers identify neurite bundles from
other lineages. Diagrams as in Fig.
2. Commissure abbreviations as in
Fig. 1; VA, ventral arch.
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Lineage 11
Lineage 11 is an Engrailed-positive cluster situated in the lateral region
of the hemineuromere. Fig.
13B-E shows an example of lineage 11 in T2. A single neurite
bundle projects dorsomedially from the neuronal cluster and runs beside bundle
19c towards the intermediate commissure. Bundle 11 splits before reaching the
commissure, with one branch (11im) extending dorsomedially and ending well
short of the midline in a spray of arbor oriented along the anteroposterior
axis (Fig. 14C,D). The other
bundle (11id) extends dorsally and ends near the termination of bundle 3id at
the point where bundle 6cd bends medially to form the pD commissure
(Fig. 13C). A small wisp of
arbor occasionally extends into the pD commissure.
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Fig. 13. Characteristics of lineage 11. (A) A thick section projection of confocal
slices showing a ventral view of the Neurotactin scaffold at the level of the
intermediate commissures; brackets identify the pI commissures. The stubby
projection of neurite bundle 11im is present in T1 and T2 but absent from T3.
(B) Ventral view of the projection a MARCM clone of lineage 11. (C-E) Thick
section projections showing the relationship of the neurite bundles from
lineage 11 to features of the Neurotactin scaffold (red) in (C) dorsal and
(D,E) intermediate neuropils. Numbers identify neurite bundles from other
lineages. Diagrams as in Fig.
2. Commissure abbreviations as in
Fig. 1.
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Fig. 14. Characteristics of lineage 12 (NB 6-1). (A) Ventral view of the projection
of a MARCM clone of lineage 12 in T3; the 12i bundles are absent. (B) Lineage
12 clone from T1. (C-F) Thick section projections showing the relationship of
the neurite bundle from lineage 12 to features of the Neurotactin scaffold
(red) in (C) dorsal, (D,E) intermediate and (F) ventral neuropils. Bundle 12c
is the posterior bundle of the posterior ventral arch; the 12i bundle
bifurcates with sub-bundles going to intermediate (12im) and dorsal (12id)
positions. Numbers identify neurite bundles from other lineages. Diagrams as
in Fig. 2. Commissure
abbreviations as in Fig. 1.
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Clones of lineage 11 were found in T2 and T1 but not in T3. Examination of
the Neurotactin-stained scaffold at the level of the intermediate commissure
shows the truncated anteromedial projections of bundle 11im in both T1 and T2
but this projection is absent from T3
(Fig. 13A). T3 also lacks the
Neurotactin bundle that corresponds to bundle 11id. In addition, T3 is has one
less Engrailed-positive cluster when compared with T1 and T2 (data not shown),
and the missing cluster is at the location of lineage 11. Consequently, we
have concluded that lineage 11 is truly missing from segment T3, rather than
being altered into producing neurons with different projection patterns.
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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Fig. 15. Characteristics of lineage 13 (NB 3-4 or 4-4). (A) Ventral view of the
projection of a MARCM clone of lineage 13. (B-D) Thick section projections
showing the relationship of the neurite bundles from lineage 13 to features of
the Neurotactin scaffold (red) in ventral neuropil. (B-D) Successively ventral
sections. Bundle 13c is one of four paired bundles in the aV tract; bundle 13i
projects to the anterior ventrolateral neuropil. Numbers identify neurite
bundles from other lineages. Diagrams as in
Fig. 2. Commissure
abbreviations as in Fig. 1.
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Lineage 14
The cluster for this lineage is situated just lateral to the lineage 4
cluster. Its single bundle of neurites (14c) contributes to the ventral
commissure and is situated anterior to the 13c bundles and immediately
posterior to the ascending bundles from lineage 2
(Fig. 16B). The bundle
projects into the ventrolateral neuropil and splays out medial to the lateral
cylinder in apparent contact with neurites from bundle 9i
(Fig. 16B,C). The projection
patterns are similar in all three thoracic neuromeres and the cluster is found
only in the thoracic neuromeres. Lineage 14 is the most medial cluster to
project through the anterior commissure to the ventrolateral neuropil. This
fits the anatomy of the cluster of local interneurons that arise from NB 4-1
in the locust (Shepherd and Laurent,
1992). Similar neurons are made by NB 4-1 in Drosophila
(Schmid et al., 1999).
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Fig. 16. Characteristics of lineage 14 (NB 4-1). (A) Ventral view of the projection
of a MARCM clone of lineage 14. (B,C) Thick section projections showing the
relationship of the neurite bundles from lineage 14 to features of the
Neurotactin scaffold (red) in ventral neuropil. Successively ventral sections.
Bundle 14c is one of four paired bundles in the aV tract. Numbers identify
neurite bundles from other lineages. Diagrams as in
Fig. 2. Commissure
abbreviations as in Fig. 1.
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Lineage 15
Lineage 15 is unique in that it appears to be composed entirely of
motoneurons (Fig. 17). The
neurites from this cluster of about 30 neurons form a single bundle (15i) that
projects dorsally through the lateral cylinder and then bends laterally where
the bundle partially defasciculates and produces some short diffuse processes
(Fig. 17B) before recollecting
together in a compact bundle that leaves the CNS through the nerve leading to
the leg imaginal discs (Fig.
17C). In the early 3rd instar lineage 15 has a neuroblast
associated with it. By the mid-to-late 3rd instar, however, the NB is no
longer evident. This is the only lineage that appears to lose its neuroblast
prior to the start of metamorphosis. Lineage 15 is found only in the thoracic
neuromeres and the projection pattern is similar in each.
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Fig. 17. Characteristics of lineage 15. (A) Ventral view of the projection of a
MARCM clone of lineage 15. (B,C) Thick section projections showing the
relationship of the neurite bundles from lineage 15 to features of the
Neurotactin scaffold (red) in (B) intermediate and (C) ventral neuropils. All
of the bundle 15 axons project into the periphery. Numbers identify neurite
bundles from other lineages. Diagrams as in
Fig. 2. Commissure
abbreviations as in Fig. 1.
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Lineage 16
The cell cluster for lineage 16 is situated along the anterior border of
the hemineuromere, just lateral to cluster 10
(Fig. 1I). The neurite bundle
(16i) projects dorsally from the cell cluster, through the lateral cylinder
between bundles 8c and 8i (Fig.
18D), and into the ventrolateral neuropil, where it makes a short,
compact projection after it emerges from the cylinder
(Fig. 18B,C). Its termination
is close to area where lineage 15 showed its partial defasciculation. This
lineage is found only in the thoracic neuromeres and has a similar projection
in each.
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Fig. 18. Characteristics of lineage 16. (A) Ventral view of the projection of a
MARCM clone of lineage 16. (B-E) Thick section projections showing the
relationship of the neurite bundles from lineage 16 to features of the
Neurotactin scaffold (red) in intermediate (B,C) and ventral (D,E) neuropils.
Numbers identify neurite bundles from other lineages. Diagrams as in
Fig. 2. Commissure
abbreviations as in Fig. 1.
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Lineage 17
This lineage is located dorsolaterally along the boundary between
neuromeres. It is Engrailed negative, so we have placed it with the anterior
lineages. Its neuroblast, along with those from lineages 18 and 9, make up the
dorsal lateral group of neuroblasts originally called the triplet
(Truman and Bate, 1988).
Fig. 19A-C shows lineage 17 in
segment A1. The neurite bundle (17i) projects into the ipsilateral
ventromedial neuropil, but then loops dorsally and reflects back at the
location of the aI commissure. We recovered only one clone of lineage 17 in
the thorax (Fig. 19D; T2). It
shows the same projection pattern as its abdominal counterpart except that
loop is more pronounced (also Fig.
19E). The 17i loops are present in T2, T3, and A1 but missing from
T1 (Fig. 19F). We have
concluded that this lineage is missing from T1, but in the absence of an
independent marker, such as the Engrailed staining we used for lineage 11, we
cannot be completely confident that the lineage is absent rather than merely
lacking its Neurotactin bundle.
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Fig. 19. Characteristics of lineage 17. (A) Ventral view of the projection of a
MARCM clone of lineage 17 in A1. (B,C) Thick section projections showing the
relationship of the neurite bundles from lineage 17 to features of the
Neurotactin scaffold (red) in intermediate neuropil (successively ventral
sections). (D) Ventral projection of lineage 17 clone in T2. In both A1 and
the thoracic neuromeres, the neurite bundle abruptly hooks dorsally before it
reaches the midline. (E) A thick transverse projection of the right
hemineuropil showing the characteristic recurved bundle from lineage 17.
Arrowhead denotes the midline. (F) A ventral view of a thick section of the
Neurotactin scaffold showing a bundle 17 in A1, T3 and T2 but missing from T1.
Numbers identify neurite bundles from other lineages; VA, ventral arch.
Diagrams as in Fig. 2. D,
dorsal commissure; I, intermediate commissures.
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Lineage 18
The cluster for lineage 18 is in the dorsolateral region of the
hemineuromere. The Neurotactin staining clearly showed that there was a
lateral lineage whose neurite bundle made up the region of the aI commissure
anterior to the 2i bundles (Fig.
20C). After crossing the midline, the bundle turns anteriorly and
inserts into a dorsal tract. We found only a single clone of this lineage
(Fig. 20A, from T2) in a CNS
that was not double-labeled for Neurotactin. Its anatomy and general location
in the neuromeres perfectly fit the prediction for this lineage based on the
Neurotactin scaffold. The 0m bundle from the median lineage terminates where
the neurite bundle (18c) from lineage 18 crosses the midline.
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Fig. 20. Characteristics of lineage 18. (A) Ventral view of the projection of a
MARCM clone of lineage 18 in T2. (B) A mid-sagittal section through the dorsal
(Dor), intermediate (Inter) and ventral (Vent) commissural bundles in T1-A1.
The neurite bundles for lineages 18 and 10 make up the region of the aI
commissure immediately anterior to the ascending bundles from lineage 2.
Bundle 18 is absent from T1 whereas bundle 10 is absent in A1. (C) A ventral
view of a thick section of the Neurotactin scaffold at the level of the
intermediate commissures. There is no lineage 18 bundle in the aI commissure
in T1, whereas it is present in the corresponding commissure in posterior
neuromeres (brackets). Numbers identify neurite bundles from other lineages.
Diagrams as in Fig. 2.
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Both horizontal (Fig. 20C)
and mid-sagittal (Fig. 20B)
sections show that the 18c bundle is missing in T1 but present in T2 through
A1. As with lineage 17, we assume that this cluster is missing from T1 but
cannot be entirely confident of its absence without an independent marker.
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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Fig. 21. Characteristics of lineage 19 (NB 7-4). (A) Ventral view of the projection
of a lineage 19 MARCM clones from T1; inset is a more ventral slice showing
bundle 19i. (B) Lineage 19 from T3 showing the contralateral (19c) and
ipsilateral (19i) bundles. (C-E) Thick section projections showing the
relationship of the neurite bundles from lineage 19 to features of the
Neurotactin scaffold (red) in intermediate (C,D) and ventral (E) neuropils.
Numbers identify neurite bundles from other lineages. Diagrams as in
Fig. 2. Commissure
abbreviations as in Fig. 1.
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Lineage 19 is present in segments T1-A1. The lineage in T3 is identical to
that in T2 but in T1 the 19c bundle is reduced to only a few fibers while the
19i bundle retains the pattern seen in more posterior thoracic lineages
(Fig. 21A). In A1, by
contrast, the 19c bundle is well developed but the 19i bundle is missing (data
not shown).
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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Fig. 22. Characteristics of lineage 20 (NB 5-4). (A) Ventral view of the projection
of a MARCM clone of lineage 20. (B-D) Thick section projections showing the
relationship of the neurite bundles from lineage 20 to features of the
Neurotactin scaffold (red) in intermediate (B) and ventral (C,D) neuropil.
Bundle 20 converges with bundles 22 and 21, and projects along the lateral
edge of the ventrolateral neuropil. It also contains 2 axons that project to
the periphery. Numbers identify neurite bundles from other lineages. Diagrams
as in Fig. 2. Commissure
abbreviations as in Fig. 1; VA,
ventral arch.
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Lineage 21
The cluster for this lineage is in the ventrolateral region of the
hemineuropil just anterior to the clusters for lineages 20 and 22
(Fig. 1I). The adult-specific
cluster is confined to the thoracic neuromeres and the projection pattern
appears identical in all of these segments. As described above, its neurite
bundle collects with the bundles from lineages 20 and 22 and projects past
bundle 1i (Fig. 23C). The
neurites then project anteromedially and terminate just posterior to the
lateral cylinder (Fig.
23B,C).
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Fig. 23. Characteristics of lineage 21. (A) Ventral view of the projection of a
MARCM clone of lineage 21. (B,C) Thick section projections showing the
relationship of the neurite bundles from lineage 21 to features of the
Neurotactin scaffold (red) in ventral neuropil. (B,C) Progressively more
ventral regions of bundle 21. Bundle 21 converges with bundles 20 and 22 but
arcs medially, when compared with the lateral arc of the other two bundles.
Numbers identify neurite bundles from other lineages. Diagrams as in
Fig. 2. VA, ventral arch.
Commissure abbreviations as in Fig.
1.
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Lineage 22
This cluster is situated between lineages 20 and 21
(Fig. 1I). The trajectory of
its neurite bundle is very similar to that of lineage 20 and terminates in the
same region of the ventrolateral neuropil
(Fig. 24B). It is sometimes
associated with a single motoneuron (Fig.
24A). It is present only in the thoracic neuromeres.
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Fig. 24. Characteristics of lineage 22. (A) Ventral view of the projection of a
MARCM clone of lineage 22. (B,C) Thick section projections showing the
relationship of the neurite bundles from lineage 22 to features of the
Neurotactin scaffold (red) in ventral neuropil. Bundle 22 converges with
bundles 20 and 21, and projects with 20 along the lateral edge of the
ventrolateral neuropil. It also contains at least one motor axon. Numbers
identify neurite bundles from other lineages. Diagrams as in
Fig. 2. Commissure
abbreviations as in Fig. 1.
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Lineage 23
This is a small, Engrailed-positive cluster in the ventrolateral region of
the neuromeres. The neurons of the cluster extend a single bundle of neurites
(23c) dorsally to the level of the pI commissure
(Fig. 25B) and insert into the
commissure at the posterior border (Fig.
25C) along with the contralateral bundle from lineage 19. The
neurites terminate soon after crossing the midline. We have had only two
clones that contained this lineage, but the Neurotactin staining suggests that
the projection pattern is the same for this clone in all of the segments in
which it resides (T1-A1). This was the only lineage that we found that also
had a glial cell as part of the clone
(Fig. 25).
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Fig. 25. Characteristics of lineage 23. (A) Ventral view of the projection of a
MARCM clone of lineage 23. (B,C) Thick section projections showing the
relationship of the neurite bundles from lineage 23 to features of the
Neurotactin scaffold (red) in intermediate (B) and ventral (C) neuropils.
Bundle 23c converges with bundles 19c (not shown) to form a posterior segment
of the pI commissure. The MARCM clone for lineage 23 has a glial cell (G)
associated with it. Diagrams as in Fig.
2. Commissure abbreviations as in
Fig. 1.
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Interrelationship of the projection patterns of lineages
The bundles of neurites from each of the lineages typically project to one
or two primary targets. The various termination points do not correspond to
any obvious glial marker and, indeed, we suspect that the targets may be
bundles from other lineages. We have numerous examples in which the neurites
from one bundle contact (e.g. Fig.
26A) or terminate in close proximity (e.g.
Fig. 26B) to the termination
sites from other bundles. The pattern of spatial overlap of lineage bundles
within the T2 neuromere is summarized in
Fig. 26C. The segmental
variation in these connections is then shown in
Fig. 27. This pattern suggests
that there are lineage-wide rules for building much of the nervous system.
This proposition is considered below.
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Fig. 26. Patterns of initial contacts amongst the neurite bundles from the 24
adult-specific lineages. (A) Confocal projection of MARCM clones showing
contact between the 14c and 9i bundles. (B) Confocal optical section showing
the neurites in bundle 1c terminating adjacent to those in bundle 22. (C)
Schematic summary of the initial contacts made by the lineages in T2. Bundles
within the pink circle terminate in the ventrolateral neuropil, those in the
blue stripe project in dorsal tracts.
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Fig. 27. Segment-specific patterns of initial contacts amongst the neurite bundles
from the adult-specific lineages. The full complement of bundles is evident in
T2. White ovals indicate which lineages or bundles are missing in other
segments. Designations as in Fig.
26.
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Discussion
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In insect embryos, the vast majority of neurons in each segmental ganglion
arise from 30 paired and one unpaired neuroblasts. In basal insect groups,
these segmental NBs show a single neurogenic period, each producing all of its
progeny during embryogenesis (Shepherd and
Bate, 1990; Truman and Ball,
1998). In insects with complete metamorphosis, however, most of
the segmental NBs in the thorax have two neurogenic periods, involving a
relatively brief phase of neurogenesis during embryonic development followed
by a much more prolonged phase during larval life
(Booker and Truman, 1987;
Truman and Bate, 1988;
Prokop and Technau, 1991).
Mapping of postembryonic NBs in the thoracic neuromeres of Drosophila
larvae indicated that 23 out of the 31 segmental NBs showed this second,
larval phase of neurogenesis (Truman and
Bate, 1988). The count from the present study is that there are 24
such clusters per hemisegment.
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;
TOP
SUMMARY
Introduction
