RESEARCH ARTICLE
53
Development 137, 53-61 (2010) doi:10.1242/dev.041749
Role of Notch signaling in establishing the hemilineages of
secondary neurons in Drosophila melanogaster
James W. Truman*,§, Wanda Moats, Janet Altman*, Elizabeth C. Marin† and Darren W. Williams‡
SUMMARY
The secondary neurons generated in the thoracic central nervous system of Drosophila arise from a hemisegmental set of 25
neuronal stem cells, the neuroblasts (NBs). Each NB undergoes repeated asymmetric divisions to produce a series of smaller
ganglion mother cells (GMCs), which typically divide once to form two daughter neurons. We find that the two daughters of the
GMC consistently have distinct fates. Using both loss-of-function and gain-of-function approaches, we examined the role of Notch
signaling in establishing neuronal fates within all of the thoracic secondary lineages. In all cases, the ‘A’ (NotchON) sibling assumes
one fate and the ‘B’ (NotchOFF) sibling assumes another, and this relationship holds throughout the neurogenic period, resulting in
two major neuronal classes: the A and B hemilineages. Apparent monotypic lineages typically result from the death of one sibling
throughout the lineage, resulting in a single, surviving hemilineage. Projection neurons are predominantly from the B
hemilineages, whereas local interneurons are typically from A hemilineages. Although sibling fate is dependent on Notch signaling,
it is not necessarily dependent on numb, a gene classically involved in biasing Notch activation. When Numb was removed at the
start of larval neurogenesis, both A and B hemilineages were still generated, but by the start of the third larval instar, the removal
of Numb resulted in all neurons assuming the A fate. The need for Numb to direct Notch signaling correlated with a decrease in NB
cell cycle time and may be a means for coping with multiple sibling pairs simultaneously undergoing fate decisions.
INTRODUCTION
How such a great diversity of cell types is generated within the
nervous system during development remains a major unresolved
question in neurobiology. In vertebrates and invertebrates, both
inductive (Briscoe, 2009; Edlund and Jessell, 1999) and lineagebased mechanisms are involved in producing this diversity (Desai
and McConnell, 2000; Cayoutte et al., 2006), but different regions
of the nervous system may be biased towards one end of this
spectrum or the other. Within insects, lineage-based mechanisms are
responsible for the vast majority of neuronal diversity, with the
possible exception of the optic lobes. In the central brain and ventral
ganglia, the neuronal stem cells (the neuroblasts, NBs) are
identifiable as individuals and each makes a characteristic set of
progeny (e.g. Bossing et al., 1996; Schmidt et al., 1997; Schmid et
al., 1999). NBs go through asymmetric, self-renewing divisions,
each resulting in a neuronal precursor cell, the ganglion mother cell
(GMC). Although there are now known to be exceptions (Bello et
al., 2008; Bowman et al., 2008; Boone and Doe, 2008), the GMC
usually undergoes a terminal division, producing two daughter
neurons. The initial progeny made by a NB are often highly diverse
and are termed the primary neurons (Hartenstein et al., 2008). Their
identities are based on the birth order of the GMCs, and this ordering
Department of Biology, Box 351800, University of Washington, Seattle, WA 98195,
USA.
*Present address: Janelia Farm Research Campus, Howard Hughes Medical Institute,
19700 Helix Drive, Ashburn, VA 20147, USA
†
Present address: Department of Biology, Bucknell University, Lewisburg, PA 17837,
USA
‡
Present address: MRC Centre for Developmental Neurobiology, King’s College
London, Guy’s Hospital Campus, London SE1 1UL, UK
§
Author for correspondence ([email protected])
Accepted 24 October 2009
is determined by a sequence of transcription factors that are passed
on to successive GMCs through time and establish neuronal fates
within a given lineage (Kambadur et al., 1998; Isshiki et al., 2001;
Grosskortenhaus et al., 2005). In the embryo, the daughter neurons
produced by the GMC division typically have distinct identities, and
this difference is controlled by Notch signaling (Spana and Doe,
1996; Skeath and Doe, 1998).
The bulk of the activity of most NBs is devoted to making
secondary neurons. The secondary neurons constitute a more
homogeneous population than the initial, primary set. In insects with
complete metamorphosis, like Drosophila, most of the secondary
neurons are born during a larval phase of neurogenesis. Studies on
the generation of secondary neurons in the caterpillar of the tobacco
hornworm, Manduca sexta (Witten and Truman, 1991), indicated
that the GMC divides to make daughters of divergent phenotypes
and that this process is then reiterated scores of times to generate two
major classes of interneurons. Similarly, in grasshoppers, Jia and
Siegler (Jia and Siegler, 2002) showed that the GMCs from the
median neuroblast in the thorax consistently produce an engrailed
positive and engrailed negative daughter, which become a local
interneuron and a projection cell, respectively. Region-specific cell
death of one sibling then sculpts the final lineage composition in a
given segment. These examples of diverse classes of cells being
generated throughout a lineage is in contrast to neurogenesis in the
mushroom bodies, where sibling neurons generated at any given
time are morphologically indistinguishable (Lee et al., 1999).
In this paper, we present a comprehensive analysis of the role of
Notch signaling in generating neuronal phenotypes within the
secondary lineages of the segmental central nervous system (CNS).
The universal pattern is for a GMC to produce two neurons of
different phenotypes, ‘A’ and ‘B’, with cell death involved in making
some lineages monotypic. A clear division of phenotype between
these A and B cell types suggest that the circuitry of the thoracic
DEVELOPMENT
KEY WORDS: Neurogenesis, Hemilineage, Notch, Numb, Neuroblasts, Drosophila
RESEARCH ARTICLE
nervous system is generated in developmental units we term
‘hemilineages’. The accompanying paper by Lin et al. (Lin et al.,
2010) shows that this pattern also holds the antennal lobes in the brain.
MATERIALS AND METHODS
Fly stocks
Flies were reared on the standard yeast-cornmeal-molasses diet. Mitotic
clones were generated using the mosaic analysis using a repressible cell
marker (MARCM) technique (Lee and Luo, 1999). We used the panneuronal driver, elavC155 GAL4 (Lin and Goodman, 1994) to obtain a range
of clones that covered all of the thoracic lineages. Wild-type clones were
generated in flies of the genotype: GAL4C155, hsFLP, UAS-mCD8::GFP;
FRT2A, tubP-GAL80/FRT2A. Notch null clones were produced using the
null allele N55e11 (Heitzler and Simpson, 1991) in the genotype:
elavC155,N55e11,FRT 19A/tub-GAL80, hs-flp, FRT 19A; UASmCD8::GFP/UAS-mCD8::GFP. Clones that showed constitutive Notch
signaling were produced by expressing the intracellular domain of Notch
[UAS-NotchCA (Larkin et al., 1996)] using the genotype: elavC155, FRT
19A/tub-GAL80, hs-flp, FRT 19A; UAS-mCD8::GFP/UAS-mCD8::GFP;
UAS-NotchCA/+. Numb activity was removed using the numb2 null allele
(Frise et al., 1996) in the combination: elavC155, UAS-mCD8::GFP, hsflp/elavC155, UAS-mCD8::GFP, hs-flp; tub-GAL80, FRT 40A/y+,numb2, ck,
FRT40A. Cell death was inhibited in homozygous clones that were mutant
for the initiator caspase dronc (Kondo et al., 2006; Williams et al., 2006)
using flies of the following genotype: hs-flp, elavC155GAL4, UASmCD8::GFP/+; tubP-GAL80, FRT 2A/droncDA8, FRT2A.
For inducing MARCM clones in recently hatched larvae, eggs were
collected over a 1- to 2-hour period, maintained at 25°C for 24 hours, and
the larvae then heat-shocked at 37°C for 45 minutes to 1 hour. Brief egg
collections were also maintained for 72 hours before heat shock to induce
clones around the start of the third larval instar.
Immunocytochemistry
Dissected nervous systems were fixed in buffered 3.7% formaldehyde for
about an hour at room temperature and then washed three times in PBS-TX
(phosphate buffered saline [pH 7.2] with 1% Triton-X100). Fixed samples
were blocked in 2% normal donkey serum (Jackson ImmunoResearch Labs,
West Grove, PA, USA) in PBS-TX for 30 minutes and then incubated in
primary antibodies for 1 to 2 days at 4°C. Primary antibodies were: 1:50 mouse
anti-Notch MAb [MAb C17.9C6 (Fehon et al., 1990)]; 1:1000 rat anti-mCD8
(Caltag Laboratories, Burlingame, CA, USA); 1:20 mouse anti-neurotactin
MAb [F4A (de la Escalera et al., 1990)]. After three to four rinses to remove
the primary antisera, tissues were incubated overnight in combinations of
FITC-conjugated and Texas Red-conjugated secondary antibodies at 1:500
dilution (Jackson ImmunoResearch Labs). Nervous systems were then rinsed,
mounted on polylysine-coated coverslips, dehydrated through an ethanol
series, cleared in xylene and mounted in DPX (Fluka, Bachs, Switzerland).
Image analysis
Confocal image stacks were typically collected at 63⫻ on either a BioRad
MRC 600 or a Zeiss 510 confocal microscope. Image stacks were processed
using Image J (http://rsb.info.nih.gov/ij/). The z-projections for a given clone
(green) included all of the sections from the cell body cluster to the end of
the neurite bundle. The z-projection for the reference channel (magenta)
typically included only the sections in the neuropil region that showed the
neurotactin-positive bundles needed for lineage identification. Images were
only globally adjusted for intensity and background.
We collected cell number data in NB clones by placing a mark on z-stack
at the center of each cell. Each cell was marked only once and a count of the
total marks yielded the total number of cells.
RESULTS
Effects of Notch on neuronal fates of the
secondary neurons of the thorax
In a given lineage, the primary neurons generated during the
embryonic phase of neurogenesis are typically diverse (Bossing et
al., 1996; Schmidt et al., 1997; Schimid et al., 1999). By contrast, the
Development 137 (1)
secondary neurons produced during the postembryonic neurogenic
phase are much more homogeneous (Truman et al., 2004; Pereanu
and Hartenstein, 2006; Brown and Truman, 2009; Zhou et al., 2009).
As seen in Fig. 1A, a NB typically generates either one or two classes
of secondary neurons, as defined by their pattern of neurite
projection. In the latter case, the two classes are based the division of
the GMC, with the two daughters showing distinct growth decisions
(Fig. 1B). This difference is then repeated for all of the GMCs
generated during the second neurogenic phase. Skeath and Doe
showed that Notch signaling is responsible for the differences in
sibling fates during GMC divisions in the embryo (Skeath and Doe,
1998), and we hypothesized that this mechanism probably also
extends into the secondary phase of neurogenesis. Immunostaining
of neuroblast clones for Notch showed the prominent membrane
localization of Notch in the NB, GMCs and an adjacent cluster of
young neurons (Fehon et al., 1991), but also, typically, two of the
young neurons had nuclear Notch (Fig. 1C,D) suggesting that these
were in the process of establishing their sibling fates. Therefore, we
examined Notch signaling in the secondary lineages using both lossof-function and gain-of-function approaches.
MARCM clones were induced postembryonically to include only
the secondary neurons born during the larval neurogenic period.
Notch loss-of-function clones were homozygous mutant for the null
allele N55e11 (Heitzler and Simpson, 1991). During Notch signaling
the receptor is cleaved and the intracellular domain translocated to
the nucleus (Struhl et al., 1993). We generated Notch gain-offunction clones either by expressing the intracellular domain of
Notch, which serves as a constitutive activator [NotchCA (Larkin et
al., 1996)], or by making MARCM clones that were null for numb,
a negative regulator of Notch (Knoblich et al., 1995; Spana and Doe,
1996). Fig. 2 summarizes the effects of manipulating Notch
signaling in the 25 secondary lineages in the ventral CNS. Nervous
systems were counterstained for neurotactin and the clones
identified by the neurotactin-positive bundle in which their neurites
project (Truman at al., 2004). As a control for the Notch loss-offunction analysis, we looked at CNSs that were counterstained for
Notch and confirmed the loss of Notch protein in the clones (data
not shown).
The cell clusters from NBs 1, 3, 6, 8, 11, 12, 13 and 19 have two
primary neurite bundles of roughly equivalent size (e.g. Fig. 3A-D).
This dichotomy was also seen in their respective GMC clones (Fig.
1B and data not shown), showing that the two neurite trajectories
reflect the different fates of the two siblings. When Notch was either
removed or constitutively activated in these lineages, we saw only
one neurite bundle, characteristic of a single sibling type, but the
bundle differed under the two conditions (Fig. 2). For example, as
seen in Fig. 3, for lineages 1, 3, 8 and 12 the Notch loss-of-function
condition resulted in the presence of the 1i, 3id, 8c and 12c neurite
bundles, respectively, whereas the 1c, 3il, 8i and 12i bundles were
present in the NotchCA clones. Relative to Notch signaling, the latter
sibling is considered the ‘A’ fate (NotchON), whereas the former
shows the ‘B’ fate (NotchOFF) (Skeath and Doe, 1998). Notch null
clones always produced a single sibling type. However, the NotchCA
clones sometimes contained one or two neurons of the ‘B’ type and
the remainder of the ‘A’ type (Fig. 3M,N). We assume the presence
of a few ‘B’ types in the NotchCA clones is because after clone
induction one or two neurons may still commit to a NotchOFF fate
before sufficient Notchintra protein is made to suppress the ‘B’
phenotype.
The shift in cellular composition of these clones was the result of
a change in cell fate and not selective cell death. This switch in fate
was clearly shown in GMC clones that lacked Notch activity. For the
DEVELOPMENT
54
Notch control of hemilineage identity
RESEARCH ARTICLE
55
Fig. 1. Relationship of sibling differences to overall lineage phenotypes. (A,B)z-projection of a lineage 6 neuroblast (A) and GMC (B) clone.
The primary neurites from the two siblings form the six contralateral intermediate (6ci) and six contralateral dorsal (6cd) bundles. The schematics
show how the primary neurites relate to the segmental commissures and leg neuropils (LNp). (C,D)The appearance of Notch immunostaining in the
nuclei of two young neurons in a lineage 1 MARCM clone. (C)A z-projection of the cell cluster. (D)An optical section showing two neurons with
nuclear Notch immunostaining; insets are magnified views of the neurons in this and the adjacent section. Yellow arrows: nuclear Notch staining.
Commissures: ad, anterior dorsal; pd, posterior dorsal; ai, anterior intermediate; pi, posterior intermediate; v, ventral.
Lineages 20, 21 and 22 were difficult to resolve. Each NB
produces one to two motoneurons as well as a large number of
interneurons that project to the leg neuropil (Truman et al., 2004).
For the Notch null clones, we saw many examples of two to four
motoneurons that were associated with a detached cell cluster
comprised of the NB, GMCs and very young neurons that lacked
neurites. We recovered no Notch null clones that contained the
interneurons for these three lineages. By contrast, we recovered
many NotchCA clones that contained the lineage 21 or 20/22
interneurons. These sometimes had an associated motoneuron, but
its presence is probably due to this cell being generated before the
production of sufficient Notchintra, as discussed above.
NB 0 makes a midline cluster of local interneurons but wild-type
clones occasionally include a projection neuron with a bifurcating
axon, the phenotype of the VUM cells produced by this neuroblast
Fig. 2. Summary of the results of removing Notch or having
constitutively active Notch on the phenotypes of NB MARCM
clones. (A)The frequency of particular lineages (for monotypic
lineages) or hemilineage bundles under the two conditions. (B)The
frequency of ‘disembodied’ lineages that included the NBs, GMCs and
a few young neurons, but few or no neurites emerging from the
cluster. The NB was identified by the location of the cluster and the lack
of its characteristic neurotactin-positive, neurite bundle. Notch null,
white bars; constitutively active Notch (NotchCA), black bars. Numbers
refer to the NBs; 20/22, combined data for lineages 20 to 22; in, local
leg interneurons, mn, motoneurons. Other designations are neurite
bundles: c, contralateral; ci, contralateral intermediate; cd, contralateral
dorsal; d, dorsal; i, ipsilateral; id, ipsilateral dorsal; ii, ipsilateral
intermediate; il, ipsilateral lateral; v, ventral.
DEVELOPMENT
above lineages, wild-type GMC clones always show the two
daughters that make different outgrowth decisions (e.g. Fig. 1B). In
Notch null GMC clones, by contrast, the two daughters were always
the same (e.g. the lineage 8 and 12 GMC clones in Fig. 3I,J). GMC
clones that expressed NotchCA typically showed both sibling types,
but for reasons stated above, we think that this is because of the
delay involved in the GAL4 system so that the daughter cells make
their fate decisions before sufficient Notchintra protein has been
made. Cell counts support the conclusion that the Notch
manipulation results in fate changes throughout a given lineage. In
the case of lineage 1, for example, control (125±10 s.e.m.; n5),
Notch null (106±4 s.e.m.; n5) and NotchCA clones (118±6 s.e.m.;
n5) had roughly the same number of neurons, despite the
differences in cell composition under the three conditions.
Neuroblasts 0, 4, 9, 14, 20, 21 and 22 also generate two classes
of neurons, but only a few of one type and many of the other. For
example, lineages 20, 21 and 22 include one or two motoneurons
as well as a large number of local interneurons that supply the leg
neuropil (Truman et al., 2004). We had previously thought that
NBs 4 and 14 produced only one type of neuron (Truman et al.,
2004), but we have since found that they produce one or two
interneurons that have trajectories that markedly differ from the
remainder of neurons in the lineage. These rare neurons are
among the first postembryonic neurons to be born and are often
missing from neuroblast clones. The effect of Notch manipulation
on these unequal lineages was also straightforward. Under one
condition, a large cell cluster containing only the major neuron
type was evident, whereas under the other condition, the minor
cell type, with numbers typically doubled, was seen along with a
slightly separated NB with its cluster of GMCs and young
neurons, but the latter apparently dying soon after their birth. For
example, NB 14 generates a large number of interneurons that
project through the ventral commissure to the contralateral leg
neuropil and one or two neurons projecting to the dorsal neuropil
(Fig. 4D). In Notch null clones, we saw only the dorsal-projecting
neurons accompanied by a detached cell cluster with the NB (Fig.
4H). In such cases, neurotactin staining showed that the lineage
14 ventral bundle was missing from that side (data not shown). In
MARCM clones expressing NotchCA, by contrast, lineage 14
clones contained only the ventral projecting neurons. The size of
the cell cluster was roughly twice that seen under wild-type
conditions.
56
RESEARCH ARTICLE
Development 137 (1)
Fig. 3. MARCM clones showing the effects of manipulating Notch signaling on lineages that produce two major classes of
interneurons. Green, anti-CD8; magenta, anti-neurotactin showing the arrangement of lineage bundles. Insets are a reduced grayscale image of
each clone. (A-D)Wild-type NB clones. (E-H)NB clones that are homozygous Notch null show a single neurite bundle. (I,J)Examples of Notch null,
GMC clones for lineages 12 and 8, showing that both siblings have the same neurite projection. (K-N)NB clones that express NotchCA. M and N
contain lineages 8 and 12 clones and the grayscale image is shown at full magnification. Both show one axon (8c, 12c) of the other sibling
phenotype. Neurite bundle names are as in Fig. 2.
neuron, whereas the other sibling probably dies.
Lineage 15 makes exclusively motoneurons that project to the leg
imaginal disc (Truman et al., 2004; Baek and Mann, 2009; Brown
and Truman, 2009; Brierley et al., 2009). Removal of Notch results
in a doubling in the size of this neuron cluster from 28±2 (n10)
cells in control clones to 57±2 (n7) neurons in Notch null clones.
Under the NotchCA condition we occasionally saw a clone with a
moderate number of motoneurons, but we were unable to
unequivocally ascribe these to lineage 15 (these neurons show very
weak expression of neurotactin). Also, under wild-type conditions,
we occasionally saw GMC clones with two lineage 15 motoneurons.
We have concluded that the majority of lineage 15 motoneurons
arise as the ‘B’ sibling, but a few may arise as part of the ‘A’ portion
of the lineage. A second, smaller motor lineage that was missed in
Truman et al. (Truman et al., 2004) is lineage 24 (Brown and
Truman, 2009), which makes the motoneurons for the proximal leg
muscles (Baek and Mann, 2009) (D. J. Brierley and D.W.W.,
unpublished). These motoneurons arise as the ‘B’ fate in lineage 24
(Fig. 2).
The role of cell death in generating monotypic
lineages
The majority of the segmental lineages are either monotypic, like
lineages 2 and 10, or make a few of one neuron type and an
abundance of the other, such as lineages 4 and 14. The Notch data
suggest that in these cases, one hemilineage survives while most or
all of the cells of the other hemilineage either die or become another
cell type, such as glia, which are not revealed by the elav driver. We
assessed the role of cell death in sculpting the composition of these
lineages by generating MARCM clones that lacked the initiator
caspase, dronc (Nedd2-like caspase – FlyBase) (Kondo et al., 2006).
DEVELOPMENT
during embryogenesis. NotchCA clones showed an expanded set of
local interneurons (Fig. 4I). Loss of Notch resulted in a compact cell
cluster around the NB, with a neurite bundle that entered the
neuropil but then dwindled away (Fig. 4E). As the neurons with the
abortive neurites are located close to the NB, we conclude that they
are recently born projection neurons that die soon after their neurite
enters the neuropil.
The remaining lineages, from NBs 2, 5, 7, 10, 15, 16, 17, 18, 23
and 24, have only a single neuron type. Clones for some of these
lineages are recovered only rarely, even under wild-type conditions,
so we can make only tentative conclusions about them. However,
others, such as NBs 2, 7, 15 and 16, appear at a high frequency. For
lineage 2 clones that lacked Notch, we saw only a cell cluster
including an NB, GMCs and young neurons without neurites at the
appropriate location, but no neurotactin-positive, neurite bundle
(Fig. 4F). The lineage 2 cluster and bundle were present in the
NotchCA clones (Fig. 4J). The opposite relationship to Notch was
seen for lineages 7 and 16, with an enlarged cluster of neurons
having the expected projection pattern under the Notch-null
condition (Fig. 2B). For lineage 7, the expression of NotchCA
resulted in the loss of the 7c neurotactin bundle and a disembodied
NB and associated cell cluster in the normal NB 7 position. NotchCA
expression in lineage 16 was complicated because this treatment
often resulted in an enlarged cell cluster because of the production
of additional NBs (see below). Neurites emerged from this cluster
and projected along the expected path but then abruptly terminated,
similar to the pattern seen in lineage 0 under the Notch null
condition. None of the neurons in the small disembodied cell
clusters expressed Broad-Z3 (Broad – FlyBase), which is a marker
of intermediate neuronal development (Zhou et al., 2009; data not
shown). This pattern suggests that one sibling survives to become a
Notch control of hemilineage identity
RESEARCH ARTICLE
57
Fig. 4. Effects of Notch manipulation on monotypic lineages. Examples of MARCM clones for lineages that are monotypic (A,B) or produce a
major and minor class of neuron (C,D). Green, anti-CD8; magenta, anti-neurotactin. Insets are a reduced grayscale image of each clone, except for
F, which shows the neurotactin channel. (A-D)Wild-type NB clones. (E-H)NB clones that are homozygous Notch null. (E)A lineage 0 clone with the
neuroblast (N) associated with a compact cell cluster containing a neurite bundle that dwindles as it enters the neuropil. (F)A lineage 2 clone
consisting of the NB and a few associated cells but no emerging neurites. The neurotactin-positive bundle of its wild-type, contralateral homolog is
indicated by the yellow arrowhead; this bundle is missing on the side with the clone. (G,H)NB clones showing just the rare cell-type and a slightly
separated NB with a small compact ball of associated cells. (I-L)NB clones that express NotchCA. Neurite bundle names are as in Fig. 2.
and 18, but for the remainder (lineages 10, 17 and 23) a new class of
interneurons appeared. Taking these results together with the Notch
data, we conclude that for the monotypic lineages one sibling
consistently lives, resulting in a single hemilineage.
Numb loss-of-function and neuronal fates
A way to bias the cells to activate Notch signaling is through the loss
of numb function (Knoblich et al., 1995; Karacavich and Doe, 2005;
Spana and Doe, 1996; Frise et al., 1996; Guo et al., 1996). However,
unlike the situation reported for the embryo (Skeath and Doe, 1998),
we found that the NBs were refractory to the loss of Numb during
early larval growth. For clones induced soon after hatching and then
Fig. 5. Examples of MARCM NB
clones that are null for the
caspase dronc. (A-E)Lineages in
which one cell type is typically
represented by only one or two
individuals. Blocking cell death results
in the addition of more neurites from
the cells of the rare phenotype (red
arrow). (A)Lineage 0, (B) lineage 4,
(C) lineage 14, (D) two examples of
lineage 21, (E) lineage 20/22.
(F-L)Monotypic lineages having one
neurite bundle (yellow arrow) acquire
a new neuronal class (red arrow)
when cell death is blocked.
(F)Lineage 5, (G) lineage 15,
(H) lineage 2, (I) lineage 10, (J) lineage
17, (K) lineage 24, (L) lineage 23.
Wild-type examples are included in
Fig. 9.
DEVELOPMENT
As summarized in Fig. 5A-E, blocking cell death resulted in a
striking increase in the numbers of the rare neuron types in lineages
0, 4, 14, 20/22 and 21. In addition, in most of the purely monotypic
lineages, we saw the appearance of neurons of novel morphology
(Fig. 5G-L). In lineage 2, the new cells also projected to the dorsal
ipsilateral neuropil, but to a more lateral extent than the normal
sibling (Fig. 5H). In the other lineages, the new cells differed
markedly from those seen in the wild-type lineage. For the two
motor lineages, the new cells were interneurons (Fig. 5G,K). By
contrast, with the blockade of cell death, lineage 5 now contained
motoneurons as well as its normal type of interneuron (Fig. 5F). We
could not resolve the effects of cell death blockade in lineages 7, 16
58
RESEARCH ARTICLE
Development 137 (1)
Fig. 6. Examples of MARCM NB clones that are
homozygous for a numb null mutation (numb2).
(A,B)Bundles from both sibling types are evident in numb
clones induced at 24 hours AEL and examined at wandering.
The lineage 1 clone (A) was among clones from other
lineages, but the 1i and 1c bundles are readily
distinguishable. (C,D)numb clones induced at 72 hours AEL
and examined at wandering showed only the A sibling in
lineage 8 (D), or only a few axons of the B type (bundle 1i)
and the remainder of the A type (bundle 1c) in lineage 1 (C).
Neurite bundle names are as in Fig. 2.
Notch and neuroblast duplication
Studies of the Drosophila brain have shown that establishing
constitutive Notch activity, either through loss of numb or by
expression of Notchintra, results in the generation of
supernumerary NBs (Bowman et al., 2008). We found that this
phenomenon also occurs in the ventral CNS but on a reduced
scale (Fig. 7). The extra NBs in MARCM clones were identified
by their large size and their expression of grainy head (Fig. 7A),
a marker for NBs (Almeida and Bray, 2005). For NB clones that
were induced around hatching and were null for Numb, 35%
(n324) showed supernumerary NBs when examined late in the
wandering stage. Unlike the massive increase in NBs seen in
some brain lineages, the increase in NBs in the ventral CNS was
modest after numb removal, with numbers of extra stem cells
ranging from one to eight. When multiple NBs were present, each
was typically associated with a set of young neurons with neurites
that showed the projection and targeting that were typical of
neurons generated at that site. The size and geometry of the cell
cluster associated with each NB suggested that the generation of
a supernumerary NB typically occurred early in the neurogenic
period. For example, Fig. 7B,C show clones for lineages 3 and 9,
which have two and three NBs, respectively. Each NB is
associated with a separate cluster of progeny and the fasciculated
bundles from each cluster join at the base of the clone where they
enter the neuropil. Such large and relatively equivalent clusters
could only arise if the NB replication occurred early in larval life.
The conclusion that NB duplication typically occurs early in
postembryonic neurogenesis is also supported by the results of
inducing numb clones at 72 hours AEL. We generated only a
limited number of such clones, but these included examples for
lineages, 8, 9, 11, 16, 19 and 20/22. For early-induced clones in
these lineages, 82% (n67) showed supernumerary NBs, whereas
only 9% (n11) of the late-induced clones had extra NBs. Overall,
only 6% (n34) of the 72-hour clones produced extra NBs.
An intriguing feature of the loss of numb function was that the
thoracic NBs differed markedly in their response to this loss. For
example, as seen Fig. 8A, lineages 0, 6 and 12 rarely, if ever, showed
a supernumerary NB, whereas lineages 9, 13 and 16 consistently
produced extra NBs. The pattern of extra NBs seen after loss of
numb was similar to that seen when the same set of NBs was
examined for their response to expression of NotchCA (Fig. 8B).
Again, lineages 0, 6 and 12 were unaffected, while 9, 13 and 16
showed multiple NBs. A lineage that responded differently to the
two treatments was lineage 19, which often showed supernumerary
NBs after loss of numb, but did not do so in seven NB clones that
expressed NotchCA.
DISCUSSION
The role of Notch signaling in establishing sibling
difference in secondary neurons and the
‘hemilineage’
The great diversity of cell types within the nervous system has
been appreciated since the studies by Cajal. Understanding the
rules that are used for generating such diversity remains one of the
Fig. 7. Effects of Notch activation on NB duplication. MARCM NB
clones showing that loss of numb (A-D) or constitutive Notch signaling
(E) results in clusters with multiple NBs. (A)Optical section through a
clone (green) showing the expression of grainy head (magenta) in the
supernumerary NBs. (B,C)Examples of lineages 3 and 9, which have
supernumerary NBs (insets); each NB is at the end of a discrete cell
cluster (arrows) and their fasciculated neurites join at the neuropil into a
common bundle that projects to their normal targets. (D,E)Examples of
lineage 13 clones that are numb null or express NotchCA, respectively.
Both treatments induced supernumerary NBs (insets). With loss of
numb (D), the 13c bundle was of normal size, whereas 13i was
hypertrophied. With NotchCA expression (E), bundle 13i was
hypertrophied but 13c was missing. N, neuroblast.
DEVELOPMENT
examined in wandering larvae, we typically saw that both types of
siblings were present (Fig. 6). Comparison of bundle size between
the two siblings was problematic, because in many cases the loss of
Numb resulted in a duplication of the NB, and this often resulted in
the disproportionate production of ‘A’ siblings (see below).
We also generated a smaller set of clones at the start of the third
instar, inducing the clones by heat shock at 72 hours after egg laying
(AEL) and examining the morphology of the resulting clones at
wandering. Lineages such as 1, 8 and 11 now showed clones that
responded as expected, with the A phenotype being the sole (Fig.
6D) or predominant (Fig. 6C) phenotype in the clone.
Fig. 8. Manipulations that cause NB duplication. Summary of the
analysis of NB MARCM clones that were (A) null for numb function or
had (B) constitutive Notch signaling. White bars, clones with a single
NB; black bars, clones with supernumerary NBs.
major goals of neurodevelopment, and the insect CNS has
contributed significantly to understanding this problem. With the
possible exception of the optic lobes, the generation of central
neurons is strictly a lineage-related process, with no regulation of
cell fates seen between lineages (Taghert et al., 1984; Witten and
Truman, 1991). In the embryo, the neurons arising from the
division of the GMC typically differ from each other (e.g. Skeath
and Doe, 1998), but for mushroom body neurons born during
larval life, the two siblings are indistinguishable (Lee et al., 1999).
The mushroom body pattern, however, is quite different from that
inferred from studies in the ventral nervous system of Manduca
(Witten and Truman, 1991) and the grasshopper (Jia and Seigler,
2002), which indicate that the two siblings assume different fates.
The data in this paper and the study on the antennal lineages by Lin
et al. (Lin et al., 2010) indicate that the mushroom body pattern is
atypical. As seen in Fig. 9, the pattern across the 25 thoracic
lineages is for the GMC to produce two different daughters. In
RESEARCH ARTICLE
59
lineages that are monotypic, a situation seemingly similar to that
seen in the mushroom body, one of the siblings is consistently
removed by programmed cell death soon after its birth.
Previous studies (Almeida and Bray, 2005) showed that Notch
was not needed for the maintenance and division of NBs during the
larval neurogenic period. Our data are completely consistent with
their findings. Notch also seems dispensable for the early
differentiation and survival of the young neurons. With Notch lossof-function, we sometimes saw an NB with a cluster of immature
cells but no neurites from maturing neurons exiting from the cluster,
but this was only seen in lineages in which the B (NotchOFF) sibling
normally dies. Similarly, expressing NotchCA also resulted in
‘disembodied’ NBs and cell clusters, but only in the lineages in
which the A (NotchON) sibling is fated for death. These disembodied
clusters were especially impressive in lineages, such as lineage 16,
that also showed supernumerary NBs due to constitutive Notch
activation. Overall, though, these data and those from the loss of the
initiator caspase Dronc, show that the abnormal death seen with
Notch manipulation is a result of the role of Notch in determining
cell fate, rather than a requirement for Notch for survival or early
differentiation.
An interesting question is whether there are global rules for fate
determination that apply across the lineages. In monotypic or almost
monotypic lineages, there is no consistent relationship of the
dominant sibling to the state of Notch signaling. In seven of the
monotypic thoracic lineages the ‘A’ sibling is the dominant surviving
cell type, whereas in nine lineages, the ‘B’ sibling is the dominant
cell type. Axonal projection, however, does correlate strongly with
whether or not the Notch pathway is activated. Only four bundles
(6ci, 7c, 18c and 19c) project into the longitudinal tracts, and these
are the B siblings of their respective lineages. Five lineages produce
motoneurons (from NBs 15, 20, 21, 22 and 24) and they also
represent the ‘B’ fate. In addition, six more lineages [0, 4, 8, 12, 13
and 19] have one sibling that has a local primary target, whereas the
other sibling projects to the periphery or across a commissure to the
contralateral side of the CNS. Lineage 4 is the only one of this group
Fig. 9. Summary of the role of
Notch signaling in the 25 lineages
of secondary neurons in the
ventral CNS. The images show the
wild-type morphology of each lineage.
The diagrams shows the path of the
neurite bundle from each hemilineage
(see Fig. 1). Green, Notch-on fate;
magenta, Notch-off fate. Open cell
bodies show cell types that do not
appear in the wild-type clones. Ones
that are known to undergo
programmed cell death are designated
by a cross. When only one cell is filled,
the oldest few neurons in the
hemilineage survive while the
remainder die.
DEVELOPMENT
Notch control of hemilineage identity
RESEARCH ARTICLE
in which the B sibling stays within its hemineuropil, whereas the A
sibling projects across a commissure. The remaining ten lineages are
uninformative because they are situations like that in lineage 3, in
which both siblings stay local, or lineage 1, in which both siblings
are projection cells. These last examples notwithstanding, there is a
strong bias for the A (NotchON) sibling to stay local and for the B
(NotchOFF) sibling to project to distant targets. Notch signaling
works through the Suppressor of hairless [Su(h)] transcription factor
(Bailey and Posakony, 1995), so one might suspect that targets
downstream of Su(h) might promote features of local interneurons
and suppress projection neuron characteristics.
Although the role of Notch in establishing sibling identity is
consistent across the lineages and through time, that of numb is
not. During embryonic neurogenesis, the loss of numb function
results in constitutive activation of Notch and the production of
only A siblings (Skeath and Doe, 1998). Early in the
postembryonic period, however, we found that the GMCs produce
daughters of both fates despite the loss of numb function (Fig.
6A,B), but by the start of the third instar numb becomes essential
for directing Notch activity (Fig. 6C,D). Therefore, early in the
secondary phase of neurogenesis, Notch signaling is not dependent
on numb, and other factors must be at play to allow Notch to
establish the differences in sibling identity. We do not know the
nature of these factors.
It may be important that the lack of a requirement for numb is
correlated with rate of division of the NB. In the embryo (CamposOrtega and Hartenstein, 1997) and in the third larval instar (Truman
and Bate, 1988) the cell cycle of the NB is less than an hour. In the
latter case, we see two or more neurons in a given cluster showing
nuclear-localized Notch (Fig. 1C,D) suggesting that siblings from
successive GMCs undergo fate decisions in an overlapping manner.
Using Numb protein at this time to bias sibling identity would ensure
that a given sibling would not be given ambiguous signals from a
cousin. When the NBs first reactivate in the second instar, however,
they are dividing much more slowly (Truman and Bate, 1988).
Assuming that the dynamics of GMC lifespan are similar to those
seen later, we suspect that at these earlier times only one sibling pair
in a cluster may be undergoing Notch-dependent decisions at a time.
This would permit the types of cell-cell interactions, such as seen in
some peripheral sensory precursor cells (Hartenstein and Posakony,
1989) or postulated for grasshopper lineages (Doe et al., 1985), to
come into play. The biasing of cells by Numb may be an adaptation
for rapid cell cycles when multiple neuron pairs are sensitive at the
same time.
Thoracic NBs show similarities to the PAN
neuroblasts
Recently, it has been shown that some NBs, the posterior asensenegative (PAN) NBs, differ from the classic scheme in that their
GMCs undergo additional divisions before making postmitotic
neurons (Bello et al., 2008; Boone and Doe, 2008; Bowman et al.,
2008). PAN neuroblasts respond dramatically to enhanced Notch
signaling; clones that either express NotchCA or are numb negative
show a dramatic expansion in the number of NBs in a given cluster,
as some of the GMCs transform into NBs (Bowman et al., 2008). In
contrast to the PAN NBs, ‘classic’ brain NBs are unaffected by
enhanced Notch activity (Bowman et al., 2008).
We also find a dichotomy in how the NBs in the ventral CNS
respond to the loss of Numb or expression of NotchCA. Most NBs
and GMCs characteristically maintain their normal pattern of
division despite constitutive Notch activation (Fig. 7). A few
lineages (NBs 8, 9, 13, 16, 17, 19 and the 20s), however, resemble
Development 137 (1)
the PAN lineages of the brain in that they respond to constitutive
Notch signaling by generating multiple NBs. Their responses are
more muted than the brain NBs, however, with only a few extra NBs
being generated in a given cluster.
As also seen for an antennal lobe lineage by Lin et al. (Lin et al.,
2010), the sensitivity to constitutive Notch signaling is greatest early
in the postembryonic life of the NB. This transient sensitivity to
Notch activation suggests that some thoracic NBs may have PAN
neuroblast characteristics early after their reactivation but later
establish a traditional mode of behavior for the remainder of their
lineage. We have not, however, found any GMC clones with more
than two siblings, which would be expected if some thoracic
lineages made a few ‘transiently amplifying’ progeny. We have not
systematically induced clones through the early period of larval
neurogenesis, however, so we cannot exclude the possibility that a
few transiently amplifying GMCs are produced among the earlier
GMCs in these lineages.
The production of supernumerary NBs has also been seen in
caterpillars of Manduca sexta, after treatment with hydroxyurea
(Truman and Booker, 1986; Witten and Truman, 1991), a drug that
blocks nucleotide reductase (Timson, 1975). Although the drug
treatment was devised to kill cycling NBs, we found that there was
a brief window at the start of postembryonic neurogenesis when
drug treatment caused some NBs to duplicate, rather than die, and
resulted in twice as many neurons of the appropriate phenotype. It
may be that some NBs in moths may also have PAN neuroblast
characteristics early in larval life and that hydroxyurea causes a
transiently amplifying precursor to cross a line that changes it into a
fully fledged NB.
It is interesting that the lineages that are the most sensitive (i.e.
show extra NBs in over 50% of the clones) to either Numb loss or
Notch activation are ones that supply local interneurons to the leg
neuropil (lineages 8, 9, 13, 14, 16, 19, 20, 21 and 22). Providing
PAN neuroblast characteristics to these NBs may be a strategy to
enhance the cell number or cellular diversity in this highly
integrative region of the CNS.
Conclusions
In this paper we present a comprehensive analysis of the role of
Notch signaling in generating neuronal phenotypes within the
secondary lineages of the thoracic ventral CNS. The universal
pattern is for a GMC to produce two neurons of different
phenotypes, A and B, with cell death involved in making some
lineages monotypic. A clear division of labor between these A and
B cell types suggest that the components of circuitry of the thoracic
nervous system are generated in developmental units we term
‘hemilineages’. We believe that viewing the construction of this part
of the nervous system through the lens of the hemilineage will allow
us to gain insights into the way by which the genome generates units
of connectivity and how these are constructed into circuits
underlying behavior.
Acknowledgements
We are grateful to S. Bray, S. Artavanis-Tsakonas, R. Fehon, J. Hirsch, Y. Hiromi,
S. Kondo, M. Piovant and H. Ruohola-Baker for fly strains and for antibodies.
Work was supported by NIH Grant NS13079 and by the Howard Hughes
Medical Institute. Deposited in PMC for release after 6 months.
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Notch control of hemilineage identity