RESEARCH ARTICLE 2027
Development 140, 2027-2038 (2013) doi:10.1242/dev.090902
© 2013. Published by The Company of Biologists Ltd
Dual role for Hox genes and Hox co-factors in conferring leg
motoneuron survival and identity in Drosophila
Myungin Baek1,*, Jonathan Enriquez2 and Richard S. Mann2,‡
SUMMARY
Adult Drosophila walk using six multi-jointed legs, each controlled by ~50 leg motoneurons (MNs). Although MNs have stereotyped
morphologies, little is known about how they are specified. Here, we describe the function of Hox genes and homothorax (hth),
which encodes a Hox co-factor, in Drosophila leg MN development. Removing either Hox or Hth function from a single neuroblast
(NB) lineage results in MN apoptosis. A single Hox gene, Antennapedia (Antp), is primarily responsible for MN survival in all three
thoracic segments. When cell death is blocked, partially penetrant axon branching errors are observed in Hox mutant MNs. When
single MNs are mutant, errors in both dendritic and axon arborizations are observed. Our data also suggest that Antp levels in postmitotic MNs are important for specifying their identities. Thus, in addition to being essential for survival, Hox and hth are required
to specify accurate MN morphologies in a level-dependent manner.
INTRODUCTION
Animal locomotion requires coordinated muscle contractions that
are controlled by motoneurons. Motoneurons receive commands in
the CNS and execute these commands by controlling muscle
contractions in the periphery. Most animals capable of movement
adopt one of two types of locomotion. Undulatory movements,
exhibited by Drosophila larvae and Caenorhabditis elegans, require
coordinated body wall muscle contractions along the dorsoventral
(DV) and anteroposterior (AP) axes. By contrast, walking by
animals such as mice and adult Drosophila requires wellcoordinated muscle contractions in each leg. Accordingly, walking
depends on the accurate specification of and connections between
the motoneurons, sensory neurons and interneurons that make up
these circuits (Dasen and Jessell, 2009; Arber, 2012).
How the exquisitely stereotyped morphologies exhibited by
neurons are established during development is an important
unsolved problem in biology. In insects such as Drosophila, neurons
are derived from neural stem cells, called neuroblasts (NBs). Each
NB is located in a stereotyped AP and mediolateral position in the
ventral nerve cord (VNC) and gives rise to a unique set of progeny
(Schmid et al., 1999; Truman et al., 2004). A series of transcription
factors expressed at sequential times during the life of each NB
provides temporal information that is integrated with spatial
information to generate neuronal identities (Pearson and Doe,
2004). NBs divide in one of two waves of activity, the first during
embryogenesis (with 30 NBs per thoracic hemisegment) and the
second during larval stages (with ~25 NBs per thoracic
hemisegment) (Truman and Bate, 1988; Truman et al., 2004).
During the embryonic wave of neurogenesis, ~17 NBs generate ~40
MNs, which target 30 larval body wall muscles in each abdominal
Departments of 1Biological Sciences and 2Biochemistry and Molecular Biophysics,
Columbia University, 701 W. 168th Street, New York, NY 10032, USA.
*Current address: Howard Hughes Medical Institute, Smilow Neuroscience Program,
Department of Physiology and Neuroscience, New York University School of
Medicine, New York, NY 10016, USA
‡
Author for correspondence ([email protected])
Accepted 4 March 2013
hemisegment (Schmid et al., 1999). During the larval wave of
neurogenesis, nine NBs give rise to ~50 MNs, which target 14
muscles in each adult leg (Baek and Mann, 2009).
In both vertebrates and invertebrates, the Hox family of
transcription factors has emerged as a group of key players in
nervous system development. Previous work on non-leg MN
Drosophila lineages demonstrated that, depending on the context,
Hox genes can regulate both apoptosis and the timing of cell cycle
exit of NBs, and apoptosis in post-mitotic neurons. For example, in
the abdomen the Hox gene abdominal A (abd-A) induces NB
apoptosis in a subset of lineages that continue to produce progeny
in thoracic segments (Bello et al., 2003; Cenci and Gould, 2005;
Maurange et al., 2008). Abdominal Hox genes also have the ability
to induce NB cell cycle exit in some abdominal lineages (Karlsson
et al., 2010). Finally, the Hox gene Abdominal-B (Abd-B) can block
or promote apoptosis of neurons, depending on the context (MiguelAliaga and Thor, 2004; Suska et al., 2011). In each of these cases,
Hox genes execute functions that diversify the nervous system along
the AP axis, analogous to their more classically defined functions in
other tissues.
In vertebrates, Hox genes have been shown to be important
regulators of MN identity in the spinal cord (Dasen et al., 2003;
Dasen et al., 2005; Jung et al., 2010). In the mouse, Hox6 and Hox10
paralogs are needed to define the lateral motor columns (LMCs)
present at brachial and lumbar levels, respectively, whereas Hox9
paralogs suppress the LMC fate at thoracic levels. Notably, the Hox
accessory factor encoded by Foxp1 gates Hox functions in a level
dependent manner, depending on the column (Dasen et al., 2008).
Within the brachial LMC, cross-regulatory interactions between 11
of the 39 mouse Hox genes establish the identities of MN pools,
providing them with their muscle-specific targeting properties in
the limb (Dasen et al., 2005). The specification of pool identities by
Hox genes is apparently distinct from their role in AP patterning,
because these cross-regulatory interactions are restricted to limbforming regions at brachial and lumbar levels of the rostral-caudal
axis.
In addition to walking, each pair of legs in Drosophila executes
segment-specific behaviors. For example, the second thoracic (T2)
legs are important for flight takeoff, whereas the T1 and T3 legs are
DEVELOPMENT
KEY WORDS: Drosophila melanogaster, Hox genes, Motoneurons, Cell death and survival, Neuroblasts
2028 RESEARCH ARTICLE
MATERIALS AND METHODS
Fly stocks
Unless otherwise noted, fly stocks were obtained from the Bloomington
Drosophila Stock Center.
yw hs-flp; ; FRT82B
yw hs-flp; ; FRT82B tubG80
yw hs-flp FRT19A
w hs-flp tubG80 FRT19A
ScrC1 AntpNS+RC3 UbxMX12 (Struhl, 1982)
yw hs-flp; FRT 82B AntpNS+RC3/MKRS
yw hs-flp tub-Gal4 UAS-GFP
FRT82B Ubx1/TM6B
FRT82B hthP2/MKRS (Sun et al., 1995)
w hs-flp FRT19A tubGal80
vGlut-Gal4 (also called OK371-Gal4) (Mahr and Aberle, 2006)
UAS-CD8GFP (on II)
UAS-p35 (on II) (from Laura Johnston, Columbia University Medical
Center, NY, USA)
vGlut-lexA (C. S. Mendes and R.S.M., unpublished)
LexO-rab3YFP (C. S. Mendes and R.S.M., unpublished)
UAS-6myc-Antp (M. A. Crickmore and R.S.M., unpublished)
UAS-AntpRNAi (Bloomington Drosophila Stock Center, stock number
27675)
Immunohistochemistry
Primary antibodies were: mouse anti-Abd-B [1:5; Developmental Studies
Hybridoma Bank (DSHB)], mouse anti-Ubx (1:20; DSHB), mouse antiAntp (8C11; DSHB), mouse anti-prospero (DSHB), mouse anti-d-CSP2
(1:200; DSHB), rat anti-Elav (1:50; DSHB), mouse anti-Repo (DSHB),
rabbit anti-Exd (Mann and Abu-Shaar, 1996), guinea pig anti-Hth (GP52)
(Ryoo and Mann, 1999), rat anti-Abd-A (from B. Gebelein, Cincinnati
Children’s Hospital Medical Center, OH, USA), rabbit anti-Scr (CLU395,
1:500) (Joshi et al., 2010), rabbit anti-Pb (e9, 1:100) (Cribbs et al., 1992),
guinea pig anti-Dfd (1:200; from W. Mcginnis, University California, San
Diego, CA, USA), guinea pig anti-Lab (1:50,000; from B. Olstein,
Columbia University Medical Center, NY, USA), guinea pig anti-Deadpan
(1:1000, from J. Skeath, Washington University in St Louis, MO, USA),
guinea pig anti-Dll (Estella et al., 2008) and rabbit anti-myc (Molecular
Probes). Secondary antibodies were: AlexaFluor488, AlexaFluor555 and
AlexaFluor647 conjugates (Molecular Probes).
CNS dissections and immunostaining were carried out as described
(Baek and Mann, 2009). Samples were incubated with primary antibodies
for two days and secondary antibodies for one day at 4°C. PBST (PBS with
0.1% Triton X-100, 0.5% BSA and 5% goat serum) was used for the
incubation and washing steps. Adult legs attached to bodies were fixed
overnight at 4°C and washed three times in PBST for 15 minutes at room
temperature. CNS and legs were dissected and mounted onto glass slides
using Vectashield mounting medium (Vector Labs).
Mosaic analysis with a repressible cell marker (MARCM) analysis
To generate MARCM clones, embryos were collected for 12 hours and
incubated for 24 hours at 25°C. First instar larvae were transferred to vials
and given heat shocks at appropriate time points. Entire lineage clones were
generated from heat-shocks at 24-36 hours after egg laying (AEL). Strains
used are listed below.
Standard MARCM clones
yw hs-flp; vGlut-Gal4 UAS-CD8GFP; FRT82B tubGal80/FRT82B,
FRT82B ScrC1AntpNs+RC3UbxMX12, FRT82B Antp NS+RC3, FRT82B Ubx1 or
FRT82B hthP2.
yw hs-flp/yw hs-flp tub-Gal4 UAS-GFP; vGlut-Gal4 UAS-CD8GFP/+;
FRT82B tubGal80/FRT82B, FRT82B ScrC1AntpNs+RC3 UbxMX12, FRT82B
Antp NS+RC3, FRT82B Ubx1 or FRT82B hthP2.
Cell death inhibition
yw hs-flp/yw hs-flp tub-Gal4 UAS-GFP; vGlut-Gal4 UAS-CD8GFP/UASp35; FRT82B tubGal80/FRT82B, FRT82B ScrC1AntpNs+RC3UbxMX12 or
FRT82B hthP2.
Compensatory axonal targeting
yw hs-flp; vGlut-Gal4 UAS-CD8GFP/vGlut-LexA lexO-rab3YFP; FRT82B
tubGal80/FRT82B, FRT82B ScrC1AntpNs+RC3UbxMX12 or FRT82B hthP2.
Ectopic Antp expression in MARCM clones
yw hs-flp FRT19A/w hs-flp tubGal80 FRT19A; +/vGlut-Gal4 UASCD8GFP; +/+ or UAS-6xmycAntp.
Knockdown of Antp levels
vGlut-Gal4, UAS-rab3YFP/+; UAS-AntpRNAi/+.
Microscopy and image analysis
Confocal images were taken as described (Baek and Mann, 2009) using the
same confocal settings for each antibody. z-stack maximum merged images
were generated and, if necessary, non-leg motoneuron clones were removed
using ImageJ.
RESULTS
Hox gene expression patterns in leg MNs
In Drosophila, there are eight Hox genes: labial (lab),
proboscipedia (pb), Deformed (Dfd), Sex combs reduced (Scr),
Antp, Ultrabithorax (Ubx), abd-A and Abd-B (McGinnis and
Krumlauf, 1992). We examined the expression pattern of the
proteins encoded by all eight Hox genes as well as hth and
extradenticle (exd), which encode Hox co-factors. As in other
tissues (Mann and Morata, 2000), nuclear Exd correlated with the
presence of Hth, so only the pattern of Hth is reported here. Around
50 leg MNs are born from nine post-embryonic NB lineages in each
thoracic hemi-segment (Baek and Mann, 2009). In the present study,
we focused on LinA (also called Lin 15) (Truman et al., 2004)
because it generates the largest number of leg MNs and can be
readily identified by its position in the VNC (Fig. 1A-C). LinA gives
rise to ~28 adult leg MNs (Baek and Mann, 2009; Truman et al.,
2010). The timing and birth order for LinA MNs are stereotyped:
LinA MNs are generated from ~50-90 hours after egg laying (AEL)
DEVELOPMENT
used for grooming the head and abdomen, respectively (Szebenyi,
1969; Kaplan and Trout, 1974; Dawkins and Dawkins, 1976;
Trimarchi and Schneiderman, 1993). These observations raise the
question of whether Hox genes play a role in diversifying MN
identities or circuitries between thoracic segments. In the work
described here, we provide evidence that during the development of
leg MNs Hox genes execute both AP patterning and segmentindependent functions. Newly born leg MNs initially express the
same Hox gene, Antennapedia (Antp), in all three thoracic segments.
Genetic analysis demonstrates that Hox genes and the gene
encoding the Hox co-factor homothorax (hth) are required for postmitotic MN survival in all three segments. By blocking cell death in
these mutants and by examining individual mutant MNs, we show
that these genes are not required to specify a leg MN identity or for
their specific lineage identity. However, these experiments reveal a
requirement for Hox genes to specify both axon and dendrite
morphologies of these MNs. Interestingly, we also find that within
a single NB lineage Antp is present at different levels that correlate
with MN birthdate, and that these levels are important for
instructing axon targeting. Thus, Hox genes are needed for both MN
survival and for specifying their morphologies in a level-dependent
manner in all three thoracic segments. Later in development, as leg
MNs differentiate during metamorphosis, their Hox expression code
changes in a segment-specific manner. The unique Hox code
exhibited by pupal stage leg MNs suggests an additional late role for
Hox genes in diversifying their properties in the different thoracic
segments, perhaps to allow them to execute segment-specific
functions.
Development 140 (9)
Hox function in Drosophila MNs
RESEARCH ARTICLE 2029
(Baek and Mann, 2009). By late third instar (~120 hours AEL),
LinA MN cell bodies reside medially in the VNC and send axons
that exit the VNC towards the leg imaginal discs through the leg
nerve (Fig. 1A-C) (Truman et al., 2004). At the pupal stage, LinA
leg MN cell bodies move to their final positions, and dendritic and
axonal arborizations begin to elaborate (Fig. 1A-C). In addition, we
found that LinA also gives rise to ~22 glial cells that end up
surrounding each thoracic neuropil in the adult VNC (Fig. 1D; data
not shown).
We found that three Hox proteins, Pb, Antp and Ubx, and both
Hox co-factors are expressed in the thoracic segments of the VNC,
where leg MN cell bodies are located, at late larval and mid-pupal
stages (supplementary material Fig. S1; Fig. S3A). Because there
are no specific markers for the leg MNs, we used mosaic analysis
with a repressible cell marker (MARCM) (Lee and Luo, 1999),
combined with the stereotyped position of LinA within the VNC to
identify this lineage unambiguously. At the third larval stage, all
LinA MNs in all three thoracic segments expressed Antp
(Fig. 2B,C,E).
In the third larval VNC, most NBs, including the LinA NB, are
positioned along the ventral side of the VNC and divide to produce
progeny in the dorsal direction (Fig. 2A). Consequently, earlier-born
neurons are located more dorsally and later-born neurons are located
more ventrally, closer to the NB (Maurange et al., 2008).
Interestingly, within single LinA MARCM clones, Antp was
detected more strongly in ventrally located cells compared with
dorsally located cells (Fig. 2B,E; supplementary material Fig. S2).
Besides Antp, Ubx was the only other Hox protein observed in LinA
MNs at the third instar stage: about three dorsally positioned LinA
MNs, and only in the T3 segment, expressed Ubx at this stage
(Fig. 2D,E). Pb, which is required for the development of the adult
proboscis, was not expressed in LinA in the third instar VNC (data
not shown).
The patterns of Hox expression changed by the mid-pupal stage
(3-4 days post-pupariation), at a time when both axon targeting and
dendritic arbors are elaborating. At this time, Antp was expressed
strongly in all LinA MNs in the T2 segment but was undetectable
in LinA MNs in T1 and T3 (Fig. 2G,J). By contrast, Ubx was
observed in all LinA MNs of the T3 segment but not in T1 or T2
(Fig. 2H-J). As in the larval stage, no other Hox proteins were
detected in LinA MNs at the mid-pupal stage. Hth and Exd were
present in all LinA MNs at both larval and pupal stages
DEVELOPMENT
Fig. 1. LinA MNs during development.
(A-C) LinA MARCM clones in T2, labeled with
vGlut-Gal4 >CD8GFP. (A) Cell bodies and
dendrites of LinA MNs at late L3, mid-pupa and
adult stages. Leg neuropil region is shaded
pink. (B) LinA MARCM clones stained for dCSP2 (red, labels the neuropil) and Dll (blue,
labels the leg imaginal discs). VNCs are
outlined with blue dotted lines. Regions shown
in C are marked with white boxes. The pupal
stage leg is outlined in pink. (C) LinA MN axons
in the periphery. Shown is the GFP channel
from the regions boxed in B. Late L3 leg
imaginal disc, pupa stage leg and adult leg are
outlined in pink. The VNC at the L3 stage is
outlined with blue dotted lines. (D) LinA
MARCM clones stained with anti-Repo (red) to
mark glia. The left-most image shows a lowmagnification view of the VNC (as depicted in
the schematic); the region boxed is shown at
higher magnification in the images to the
right. About 22 LinA-derived glia are visualized;
these surround the neuropil in the adult (data
not shown).
2030 RESEARCH ARTICLE
Development 140 (9)
(supplementary material Fig. S3A). In the adult, these segmentspecific Hox expression patterns were maintained (supplementary
material Fig. S3C-F). In summary, Hox expression in LinA is
dynamic: in late third instar VNCs, all LinA MNs express Antp
regardless of the thoracic segment (Fig. 2E). At this stage, the levels
of Antp correlate with the MN birthdate within LinA: older MNs
have lower levels of Antp than do younger MNs. Approximately
72 hours later, during metamorphosis, LinA MNs in T1 express no
Hox genes, those in T2 express Antp and those in T3 express Ubx
(Fig. 2J).
Hox genes and hth are required for MN survival
To determine the function of Hox genes and hth in LinA, we used
the MARCM method to genetically remove their activities from this
lineage. To ensure that we were examining the loss of all thoracic
Hox function, most experiments used the triple-mutant chromosome
Scr Antp Ubx, although, as described below, our key findings were
confirmed with single Hox mutant chromosomes. Depending on the
experiment, MARCM clones were labeled with mCD8GFP driven
by either vGlut-Gal4, which is a post-mitotic MN driver, or by
combining tub-Gal4, a ubiquitous driver, with vGlut-Gal4. The
ubiquitous Gal4 driver was added to ensure that we were monitoring
all LinA progeny, not just those that expressed vGlut-Gal4.
In either Scr Antp Ubx or hth mutant clones, the number of LinAderived neurons (Elav+) was dramatically reduced when examined at
the late third instar larval stage (~11 compared with 35 neurons in
wild type) or in the adult [about four (hth) or nine (Scr Antp Ubx)
compared with 25 MNs in wild type] (Fig. 3A,C,E,G; supplementary
DEVELOPMENT
Fig. 2. Hox expression patterns. (A) Schematic of LinA in the larval VNC. The LinA NB (light blue) is positioned ventrally in the VNC and its progeny
(green) extend dorsally. Consequently, earlier born MNs are located more dorsally and later born MNs are located more ventrally. Planes 1 to 5 refer to
the z-stack slices shown in B-D. (F) Schematic of LinA in a mid pupa (3-4 day APF) VNC. LinA MN cell bodies (green) are located ventrally. Planes 1 to 5
represent the z-stack slices in G-I. (B-D,G-I) LinA MARCM clones in late L3 (~120 hour AEL; B-D) and at the mid-pupa stage (G-I) showing five z-stack
slices from ventral (1) to dorsal (5). Magnified areas are indicated with the yellow arrows of clones in the low-magnification images on the left and
regions examined are depicted in the schematics. (B) LinA clones in T1 (top row), T2 (middle row) and T3 (bottom row) stained for Antp (red). LinA
clones in T2 and T3 were generated in a single sample. (C) Antp−/− LinA clone in T3 segment during late larval stages stained for Antp (red); the absence
of staining in the clone confirms the specificity of the signal seen in B. (D) LinA clones in T1 and T3 (in a single sample) stained for Ubx (red) and Scr
(blue). Approximately three cells in the LinA clone in T3 express Ubx. (G) Antp (red) is expressed strongly in all T2 LinA MNs, but not in T1 or in T3. Antp
levels do not correlate with their dorsal-ventral position at this stage. (H) LinA clones in T1 and T3 stained for Ubx (red) and Scr (blue). Ubx is expressed
in all LinA MNs in T3. Most MNs express high levels of Ubx. Scr is not expressed in LinA MNs. (I) Ubx−/− LinA clone (green) in T3 segment stained for Ubx;
the lack of staining confirms the specificity of the signal seen in H. (E) Summary of Hox expression patterns in LinA MN progeny in late L3. (J) Summary
of Hox expression patterns at the mid-pupa stage. In T2, but not T1 or T3, Antp is expressed in all LinA MNs, mostly at high levels. In T3, Ubx is expressed
in all LinA MNs. Hth is expressed in all LinA MNs in all three thoracic segments in both larval and pupal stages but its levels vary (supplementary material
Fig. S3A).
Hox function in Drosophila MNs
RESEARCH ARTICLE 2031
Fig. 3. Hox and hth are required to generate the correct number of LinA progeny. (A-F) LinA MARCM clones labeled with tub-Gal4; vGlut-Gal4
>CD8GFP (green, outlined with light blue dotted lines), stained for Elav (red, marks mature neurons) and Pros (blue, marks young neurons). Magnified
areas are indicated by boxes and yellow arrows and the position of clones in the VNC are shown by the red boxes in the schematics on the left. (A) WT,
(B) WT +p35, (C) Scr−/−Antp−/−Ubx−/−, (D) Scr−/−Antp−/−Ubx−/− +p35, (E) hthP2, (F) hthP2 +p35. (G,H) Quantification of neurons (G; Elav+) and glia (H;
identified by position and the lack of Elav staining; see Fig. 1D) in individual LinA clones at late L3 (~120 hour AEL). Each point represents the cell
number for a single MARCM clone. Ectopic p35 expression rescued the number of neurons (G) but not the number of glia (H). Compared with wild-type
LinA clones, about ten additional neurons were generated when p35 was expressed, suggesting that apoptosis normally eliminates these cells (Truman
et al., 2010). Elav– (non-glia) cell numbers were not significantly changed in mutant LinA clones [median values: WT (8); WT +p35 (3); Scr−/−Antp−/−Ubx−/−
(4); Scr−/−Antp−/−Ubx−/− +p35 (8); hthP2 (4); hthP2 +p35 (2)]. Black bars represent median values. (See also supplementary material Fig. S4A.)
Antp in Ubx mutant clones in this segment and a functional
redundancy of these two genes for promoting survival. Consistent
with this notion, when examined at the pupal stage, we observed
Antp protein in Ubx mutant T3 LinA clones (supplementary
material Fig. S4B).
Neuronal apoptosis in Hox and hth mutant LinA
clones
The reduced number of MNs observed in Hox or hth mutant LinA
clones could be caused by several mechanisms, which are not
mutually exclusive: for example, early NB cell cycle exit, premature
NB cell death, or post-mitotic MN cell death. To distinguish
between these mechanisms, we first checked whether the LinA NB
undergoes premature cell death. At the early third instar larva stage,
DEVELOPMENT
material Fig. S4A). These phenotypes were observed in all three
thoracic segments. Because these experiments included the ubiquitous
driver tub-Gal4, they argue against the possibility that MNs are being
transformed to non-MN cell types in Hox or hth mutants.
Antp single mutant LinA clones also resulted in a reduced number
of MNs in T1 and T2 but, curiously, not in T3 (in the adult, about
nine LinA MNs were observed in T1 and T2 segments and ~25
neurons in T3) (supplementary material Fig. S4A). Ubx mutant
LinA clones also did not show a reduction in MN number in T3: in
the adult, ~25 MNs were observed in Ubx mutant LinA clones in all
three segments (supplementary material Fig. S4A). We
hypothesized that the difference in the phenotypes observed in T3
for Scr Antp Ubx clones (about nine MNs) and the singly mutant
Antp or Ubx clones (~25 MNs) might be due to de-repression of
2032 RESEARCH ARTICLE
Development 140 (9)
Fig. 4. Axon targeting and dendritic defects in Hox and
hth mutant LinA MNs. All panels show axons in adult T2 legs
and dendrites in the VNC of LinA MARCM clones. In the VNC,
the leg neuropil and midline are marked by blue dotted
circles and red dotted lines, respectively. See supplementary
material Fig. S5A,B for examples in T1 and T3, and
supplementary material Fig. S5D for a summary. (A) LinA MNs
projecting to T2 legs. Axon targeting defects are marked with
red lines. Wild-type and Hox mutant LinA clones have thin
dendrites that cross the midline (blue arrows); these are
missing in hth LinA clones (red arrow). (B) LinA MNs
expressing p35 projecting to T2 legs. Ectopic expression of
p35 partially rescues the axon targeting phenotypes for both
Hox and hth mutants. hth mutant LinA clones lack dendrites
crossing the midline (red arrow).
surrounding the leg neuropil (Fig. 1D; data not shown). As with
MNs, the number of glia was strongly reduced in Hox or hth mutant
LinA clones (Fig. 3H). Surprisingly, however, although ectopic p35
expression in mutant LinA clones rescued the number of MNs, the
number of glia was not rescued (Fig. 3H), arguing that apoptosis is
not the cause of the reduced number of glia in Hox or hth mutant
LinA clones. With the exception of very low levels of Antp, we also
did not detect expression of Hox genes, hth or exd in LinA-derived
glia (supplementary material Fig. S3B). Taken together, these data
suggest that Hox and Hox co-factors might be required in the LinA
NB to specify the glia cell fate.
Defects in axon targeting of Hox and hth mutant
leg MNs
To determine whether, in addition to survival, Hox genes and hth are
required for MN identities, we examined both axon targeting and
dendritic arbors of mutant leg MN clones. Wild-type LinA MNs
mainly target the femur and tibia and have a complex dendritic
morphology in the neuropil (Fig. 4A) (Baek and Mann, 2009;
Brierley et al., 2009; Brown and Truman, 2009). In either Hox (Scr
Antp Ubx) or hth mutant LinA clones, the surviving MN axons
targeted the same region of the leg as did wild-type LinA clones.
Compared with wild-type clones, fewer branches were observed,
consistent with the fewer number of cells (Fig. 4A shows examples
in T2; see supplementary material Fig. S5A,B for examples in T1
and T3). The distal region of the tibia was most highly affected;
other axon branching defects appeared to be randomly distributed
within the LinA-targeted region of the leg (summarized in
supplementary material Fig. S5D). Although axon branching errors
were not observed in Ubx mutant LinA clones, Antp mutant LinA
DEVELOPMENT
NBs, labeled by Deadpan (Dpn) (Lee et al., 2006), were observed
in both wild-type and Hox mutant LinA clones (supplementary
material Fig. S4C-F). At this time point, the LinA NB was
associated with a similar number (~18) of neuronal progeny (as
assessed by Elav+ staining) in both wild-type and mutant LinA
clones (supplementary material Fig. S4C-F). These data argue
against the idea that either premature LinA NB cell death or early
cell cycle exit account for the lower number of LinA MNs. To
determine whether apoptosis is responsible for the reduced number
of MNs, we blocked cell death by expressing the caspase inhibitor
p35 (Hay et al., 1995). When examined at the late third instar stage,
the expression of p35 in Hox or hth mutant LinA clones
(simultaneously using tub-Gal4 and vGlut-Gal4) rescued the
reduced number of MNs (Fig. 3D,F,G). In addition, wild-type or
mutant LinA clones expressing p35 generated a greater number of
neurons than did wild-type LinA clones not expressing p35 (from
~35 to 45 cells; Fig. 3B,G). These findings are consistent with
previous observations showing that a subset of LinA progeny
normally undergoes apoptosis (Truman et al., 2010). An alternative
explanation is that the LinA NB, which normally disappears at the
mid-third instar stage (Truman et al., 2004), is kept alive by p35.
When expression of p35 was limited to post-mitotic MNs (by using
only vGlut-Gal4) we observed only a partial rescue (data not
shown), suggesting that both mechanisms might be operational.
However, we cannot rule out the possibility that p35 levels are too
slow to accumulate in time to efficiently rescue MN survival, in part
owing to perdurance of Gal80 in these MARCM clones.
In the MARCM experiments using tub-Gal4, which labels both
MNs and glia, we found that the wild-type LinA NB also gives rise
to ~22 glial cells (as assessed by anti-Repo staining) that end up
Hox function in Drosophila MNs
RESEARCH ARTICLE 2033
clones displayed targeting defects in all three segments, including
T3, despite no affect on MN cell number in this segment
(supplementary material Fig. S5C). These results argue that Hox
genes, and Antp in particular, play a crucial role in specifying MN
morphologies in addition to maintaining their survival. Although
the dendritic phenotype is more difficult to assess owing to the large
number of MNs in a LinA clone, some defects could be observed.
Most clearly, we found that a small number of midline-crossing
dendrites were absent in hth mutant LinA clones, in all three
thoracic segments (Fig. 4A; supplementary material Fig. S5A,B).
This phenotype was not observed in Hox mutant clones (Fig. 4A;
supplementary material Fig. S5A,B).
To explore further the idea that Hox genes are required for
conferring MN identities, we examined the phenotypes of Hox
mutant LinA-derived MNs in which survival was rescued by the
expression of p35. p35 expression in otherwise wild-type LinA
clones generated MNs with axon-targeting properties very similar
to those of wild-type LinA clones (Fig. 4B). Hox mutant axons that
were rescued by p35 also targeted the same region along the
proximodistal axis of the leg as did wild-type LinA MNs (Fig. 4B
for examples in T2; supplementary material Fig. S5A,B for
examples in T1 and T3). However, most samples exhibited some
targeting defects (summarized in supplementary material Fig. S5D).
These errors were not limited to one region of the LinA-targeted
leg and typically consisted of an absence of terminal arborizations.
In the neuropil, hth; p35+ clones exhibited a similar midlinecrossing defect as seen in hth clones not rescued by p35 (Fig. 4B;
supplementary material Fig. S5A,B). Together, these data
demonstrate that in the absence of Hox or hth inputs LinA MNs are
not appropriately specified, even when death is blocked. However,
they also show that in this lineage Hox genes and hth are not
required for forming leg MNs and for specifying a LinA identity.
Removing Hox functions from individual MNs
results in axon- and dendritic-patterning errors
The experiments described above examined entire LinA clones,
each labeling >25 MNs. Consequently, these experiments do not
have cellular resolution. To examine the role of Hox genes and hth
DEVELOPMENT
Fig. 5. Aberrant axon branching and dendritic
morphologies in individual Hox or hth mutant
MNs. Examples of single-cell MARCM clones. For
each clone, both axon and dendrites are shown.
The neuropil and midline are marked by blue
dotted circles and red dotted lines, respectively.
(A) Removing Hox and hth functions results in
distinct phenotypes. Dendritic and axon defects
are indicated by blue arrowheads and red lines,
respectively. Coxa- and femur-targeting MNs are
from LinB; the tibia-targeting MN is from LinA.
(B,C) Examples of mutant single MNs in which
there is a mismatch between axon and dendrite
morphologies. The left-hand images show wildtype MNs, each outlined in red or blue. The righthand images show Hox (B) or hth (C) mutant MNs
that share either the axon or dendrite
morphologies with one of the two wild-type MNs
as indicated. (See also supplementary material
Table S1.)
2034 RESEARCH ARTICLE
Development 140 (9)
Fig. 6. The levels of Antp are instructive for MN
targeting. (A) Antp levels are highest in late-born
LinA progeny and lowest in early-born LinA
progeny, and early-born MNs target proximal
regions of the leg segments, whereas late-born MNs
target distal regions (Baek and Mann, 2009).
(B) Examples in T2 (left) and T3 (right) of LinA MNs
expressing either GFP (top row) or Antp (bottom
row) via the vGlut-Gal4 driver. Pink and blue boxes
highlight the proximal femur (PF) and distal femur
(DF), which show a decrease and increase in
branching, respectively. Defective midline-crossing
dendrites are marked by light-blue arrowheads. (See
also supplementary material Fig. S6.)
Different levels of Antp are instructive for MN
morphologies
One of the intriguing observations described above is that Antp
levels vary among LinA progeny such that younger MNs express
higher levels than older MNs (Fig. 2B; supplementary material Fig.
S2). In our previous analysis of this lineage (Baek and Mann, 2009),
we found that older MNs from LinA had a tendency to target the
proximal femur and proximal tibia, whereas younger MNs targeted
the distal regions of these same segments. To test whether Antp
levels play a role in this targeting bias, we generated LinA MARCM
clones in which Antp was ectopically expressed via the post-mitotic
driver vGlut-Gal4. This manipulation did not affect MN survival or
final cell number (data not shown). However, we found that there
was a clear reduction of axon branches in the proximal femur and
increased branching in the distal femur (Fig. 6; supplementary
material Fig. S6). Weaker effects in the same direction were
observed in the tibia. In a separate experiment, we knocked down
Antp levels in all post-mitotic MNs by driving Antp-RNAi using
vGlut-Gal4 and observed a distal-to-proximal shift in axon targeting
(supplementary material Fig. S7). Together, these observations
suggest that high levels of Antp, normally present in younger LinA
progeny, promote the identity of MNs that target the distal regions
of the femur and tibia. Conversely, MNs born earlier in the lineage
target the proximal regions of these segments, in part owing to lower
levels of Antp.
No compensatory targeting from MNs from other
lineages
In all of the experiments described above, manipulating the activity
of Hox or hth did not affect the MN or LinA identity of progeny
cells. For example, cells derived from Hox mutant LinA and rescued
by p35 expression remained MNs and targeted the same region of
the leg as did wild-type LinA MNs. These observations suggest that
the identities of progeny MNs are hardwired according to the NB
lineage from which they are derived. As an additional test of this
idea, we asked whether MNs derived from other lineages might be
able to take the place of MNs that die in the absence of Hox or hth.
To answer this question, we generated positively labeled Hox or hth
mutant NB clones in a background in which all MNs were labeled
by the expression of rab3-YFP (Zhang et al., 2007). In this
background, we generated Hox and hth mutant LinA and LinB
MARCM clones, labeled by mCD8GFP driven by vGlut-Gal4
(Fig. 7A). Axons and neuromuscular junctions (NMJs) in mutant
MN clones were compared with those in wild-type MNs. For both
LinA and LinB, legs innervated by either Hox or hth mutant clones
DEVELOPMENT
in individual LinA MNs, we changed the MARCM protocol to
generate individual MNs that are mutant for these genes (Baek and
Mann, 2009). Altogether, we were able to recover about two-thirds
of the MNs generated by LinA, suggesting that most LinA MNs can
survive when the functions of these genes are removed at this last
cell division. Moreover, more than half of these mutant MNs
showed an aberrant morphology, although with variable penetrance
(supplementary material Table S1). For many Hox or hth mutant
MNs, dendritic or axonal arborization defects were observed,
although the requirement for these factors often differed (Fig. 5;
supplementary material Table S1). For example, a coxa-targeted
MN had ectopic dendrites when mutant for hth, but not Hox
(Fig. 5A, top row), whereas a tibia-targeted MN had ectopic
dendrites when mutant for Hox, but not hth (Fig. 5A, bottom row).
In both of these examples, axon branching appeared normal. By
contrast, a femur-targeted MN had fewer axon branches when either
Hox or hth functions were removed, but ectopic dendrites were only
observed when the MN was mutant for hth (Fig. 5A, middle row).
From these data it appears that individual MNs often have a unique
requirement for hth and Hox functions.
In most cases, individual MNs mutant for hth or Hox exhibited
defective, as opposed to transformed, dendritic morphologies and
axon branches. However, in two examples (out of 125 total single
MN clones), we observed MNs that appeared as though they were
hybrids of two different MNs. In these cases, the mutant MNs had
dendritic and axon morphologies that are usually observed in different
wild-type MNs, suggesting that the identities of these MNs were
partially transformed (Fig. 5B,C). For example, Fig. 5B shows a Hox
mutant MN that targets the distal femur but has a dendritic
morphology normally found in a MN that targets the proximal tibia.
Fig. 5C shows an hth mutant MN that targets the distal femur but has
a dendritic morphology normally found in a MN that targets the
proximal tibia. Although they are rare, these examples suggest that
separable genetic programs control dendritic and axonal
morphologies, and that Hox and hth often execute distinct functions
at the single-cell level.
Hox function in Drosophila MNs
RESEARCH ARTICLE 2035
had regions that remained unlabeled by both rab3-YFP and GFP
(Fig. 7B,C). We conclude that, despite the lower number of MNs
produced by these mutant lineages, there was no compensatory
targeting by wild-type MNs from wild-type non-LinA lineages.
Thus, even when many muscle targets are available, leg MNs
apparently maintain their lineage-derived identity.
DISCUSSION
In addition to their well-documented role in specifying differences
along the AP axis during animal development (McGinnis and
Krumlauf, 1992), Hox genes also set up differences along the AP
axis within the nervous system. In Drosophila, some NB lineages
produce more or less progeny depending on where they are along
the AP axis and, therefore, which Hox gene they express (Bello et
al., 2003; Maurange et al., 2008). In the vertebrate nervous system,
sub-regions of the hindbrain, known as rhombomeres, obtain their
identities as a result of which Hox genes are expressed (McGinnis
and Krumlauf, 1992). And, more recently, Hoxc9 has been shown
to play a crucial role in specifying thoracic as opposed to limbinnervating motoneurons in the mouse spinal cord (Jung et al.,
2010). In the work described here, we present evidence for a Hox
function that is independent of AP patterning: the survival of postmitotic MNs in the Drosophila thorax. Interestingly, in all three
thoracic segments this function appears to be carried out primarily
by Antp, although in T3 either Antp or Ubx can apparently execute
this function. Moreover, for MN survival, similar (though not
identical) phenotypes were observed for Hox and hth mutant LinA
clones, suggesting that for this function, Hox and hth might function
together to regulate the same target genes, as they do in other
circumstances. Because our experiments depend on clonal analysis,
our results also demonstrate that Hox genes are required
autonomously within a single NB lineage for keeping motoneurons
alive. Although this could represent a cell-autonomous function
within individual MNs, we cannot exclude the possibility that the
reduced number of LinA-derived glia in mutant LinA clones
contributes to the poor survival of MNs.
DEVELOPMENT
Fig. 7. No compensatory targeting in
legs with Hox or hth mutant LinA
clones. (A) Experimental design. In this
experiment, Hox or hth mutant (M*) Lin
A MARCM clones (labeled by vGlutGal4; UAS-CD8GFP) were generated in a
background in which all MNs were
labeled by vGlut-LexA; LexO-rab3YFP.
(B,C) Examples of Hox mutant clones
(B) and hth clones (C) in LinA and LinB.
For comparison, the wild-type (WT)
contralateral legs from the same
animals are shown on the right.
Targeting defects are indicated by red
lines and are still observed when all
MNs are visualized (YFP+GFP).
In vertebrates, MN survival is typically dependent upon the
reception of and retrograde signaling by neurotrophic factors
derived from peripheral tissues (Zweifel et al., 2005; Kanning et al.,
2010). By contrast, in Drosophila and other insects, MN survival is
independent of the target muscle (Goodman and Bate, 1981;
Whitington et al., 1982; Nässel et al., 1986; deLapeyrière and
Henderson, 1997). Nevertheless, a family of neurotrophins that
mediate neuronal survival has been identified in Drosophila that
acts through Toll-like receptors (Zhu et al., 2008). Based on our
results, we speculate that Hox and hth are required for the reception
or production of these neurotrophins in LinA.
Although the number of MNs that die in these mutant
backgrounds was consistent from sample to sample, the neurons
that were affected were apparently randomly distributed within the
lineage. One possible explanation for this observation is that without
Hox or hth inputs the identities of surviving MNs become
randomized, resulting in random targeting. However, our single-cell
clone analysis argues against this idea, as MN identities remained
largely intact despite exhibiting aberrant morphologies.
Alternatively, we favor the idea that removing Hox or hth results in
partial failure in the reception of survival factors, resulting in
stochastic and partially penetrant cell death among all the MNs
within the lineage.
MN identities in the absence of Hox input
In addition to promoting MN survival, our data also reveal a
requirement for Hox gene functions in the specification of
motoneuron morphologies. When entire LinA lineages were mutant
for either Hox or hth, and MN survival rescued by p35, LinA MNs
targeted the same positions along the proximodistal (PD) axis as did
wild-type LinA MNs; axons targeting to ectopic positions along the
leg were not observed. These observations suggest that Hox or hth
inputs are not required for this level of MN specification; the cells
remain MNs and their ‘LinA identity’ remains intact. However,
within the correctly targeted region, mutant LinA MNs exhibited
frequent axon-targeting defects. These defects were not limited to a
specific subregion of the LinA-targeted domain, suggesting that no
specific subset of LinA MNs has an absolute requirement for these
functions.
The maintenance of a LinA identity by Hox or hth mutant
lineages is reminiscent of the observation in vertebrates that the
targeting of limb-level MNs appears superficially normal in the
FoxP1 mutant (Dasen et al., 2008; Rousso et al., 2008). In this case,
targeting defects were only revealed when individual MN pools
were labeled by backfilling from specific muscles. Our single-cell
mutant analysis argues against a similar scrambling of identity
within LinA occurring in Hox or hth mutant MNs. Out of 87
individual Hox mutant LinA neurons that were analyzed (distributed
across all three thoracic segments), 55 had a non-wild-type
morphology. However, despite clear defects, these MNs did not
appear to have a transformed identity. Instead, most mutant MNs
continued to approach the same muscle target and either exhibited
an aberrant dendritic (44/87) or axon branching (18/87)
morphology. Moreover, for many LinA MNs that showed aberrant
phenotypes when mutant, the same MNs had wild-type
morphologies in other samples (supplementary material Table S1).
One explanation to account for these partially penetrant phenotypes
is that they result from the perdurance of the Hox or hth gene
products following clone induction. It is also feasible that removing
only a single component from potentially large transcription factor
complexes has only a limited effect because of multiple, partially
redundant inputs. Answering this question will ultimately require
Development 140 (9)
the identification and characterization of target genes that are
directly regulated by Hox and hth in developing MNs.
The differences between the role of Hox genes in Drosophila
compared with their role in specifying MN identities in vertebrates
might be a consequence of the additional requirement for
establishing motoneuron pools in vertebrates, which, for forelimb
MNs, depends on at least 11 different Hox gene products (Dasen et
al., 2005). By contrast, the relatively lower number of motoneurons
innervating the muscles of the Drosophila leg might not require
such a complex combinatorial transcription factor code. We
speculate that the expansion of Hox gene inputs governing MN
development in vertebrates, itself a consequence of a large increase
in Hox gene number, might have enabled the evolution of more
complex limbs and musculature.
Different requirements for Hox and Hox co-factor
inputs
As noted above, we found that both Hox and hth inputs are similarly
required for MN survival, suggesting that these factors might
function as co-regulators of the same target genes, as has been
observed for other Hox functions (Rieckhof et al., 1997; Mann and
Affolter, 1998). However, other phenotypes were different when
Hox or hth functions were removed (Figs 4, 5). For example, for a
femur-targeting MN we found that Hox inputs were required for
accurate axonal targeting, whereas hth was required to block the
aberrant growth of dendrites towards the midline in the CNS
(Fig. 5). By contrast, for a tibia MN, we found that Hox functions
were required to block aberrant dendrite growth, whereas hth
appeared to play no role in either dendrite or axon morphology
(Fig. 5). In a third example, hth was required for a wild-type
dendritic pattern of a coxa-targeting MN, whereas Hox functions
apparently played no role in this MN (Fig. 5). Taken together, these
data support the idea that independent genetic programs control
dendritic patterning and axonal patterning. In addition, for these
aspects of MN morphology it appears that Hox proteins do not use
Hth (and probably also not Exd) as a co-factor. Instead, these data
suggest that Hox and hth are regulating different sets of target genes
that independently affect dendritic or axonal targeting, depending on
the MN. Previous work suggests that, for specifying dendrite
morphologies, one pathway that may be targeted is the Robo/Slit
pathway (Brierley et al., 2009).
Antp levels as a timing mechanism
The progeny of individual NBs in Drosophila differ in part owing
to their birthdates within their respective lineages. For several NB
lineages, this temporal information is controlled by a series of
sequentially expressed transcription factors (Pearson and Doe,
2004). Here, we provide evidence that another mechanism to
diversify cell fates within a lineage is by varying the levels of the
Hox transcription factor Antp. The experiments supporting this
conclusion were clearest when we ectopically expressed Antp in
post-mitotic progeny using the vGlut-Gal4 driver. In these
experiments, we observed a clear shift from MNs targeting the
proximal femur, which are born early in the lineage, to distally
targeting MNs, which are born later in the lineage. Conversely,
knockdown of Antp levels in post-mitotic MNs by RNAi resulted
in a distal-to-proximal shift in axon targeting. In contrast to these
knockdown experiments, when we eliminate Hox gene expression
using null alleles we found that both distally and proximally
targeting MNs were affected. Based on these findings, we conclude
that low levels of Antp results in a phenotype that is distinct from
that observed in the absence of Antp. The data suggest that low Antp
DEVELOPMENT
2036 RESEARCH ARTICLE
levels (normally present in early-born MNs) instruct MNs to target
distal muscles, whereas MNs with no Antp show neither a proximal
nor a distal bias in their axon targeting.
Although to our knowledge this mechanism has not been
previously observed for a Hox protein, the levels of the BTB/zinc
finger transcription factor Chinmo are instructive in NB lineages in
the Drosophila mushroom body (Zhu et al., 2006). In this case,
Chinmo levels are controlled translationally, by sequences in the
5⬘UTR of its mRNA. Although such a mechanism may account for
varying Antp levels in LinA, it is also possible that Antp is
continuously expressed in the LinA NB, but not in its progeny,
resulting in its gradual decline in post-mitotic neurons. Leveldependent functions have also been observed for the Hox accessory
protein FoxP1 in vertebrate MNs and for Hth in Drosophila
embryonic lineages (Dasen et al., 2008; Karlsson et al., 2010).
However, these two cases are distinct from the Chinmo and Antp
examples, which influence cell fates within individual NB lineages.
By contrast, in the case of FoxP1, its levels are important for
establishing differences between MN columns: FoxP1 levels are
high in limb-level LMCs, but low in intervening MNs that project
to thoracic body wall muscles. In the case of Hth, an abrupt increase
in levels during embryogenesis is important for accurate gene
regulation in the progeny of NB 5-6 in the Drosophila thorax.
Nevertheless, the findings presented here reinforce the idea that
transcription factor levels play an important role in their function,
and in conferring temporal information to NB lineages.
Acknowledgements
We thank G. Struhl, J. Skeath, C. Doe, L. Johnston, B. Ohlstein, W. McGinnis,
B. Gebelein, B. McCabe, C. Mendes, Hybridoma Bank, Bloomington
Drosophila Stock Center for materials; and B. Noro, W. Grueber and J. Dasen
for comments on the manuscript.
Funding
M.B was partially supported by a Gatsby studentship (The Gatsby Initiative in
Brain Circuitry). The project was funded by grants from Project A.L.S [awarded
to R.S.M.]; and the National Institutes of Health [grant number R01NS070644
to R.S.M.]. Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.090902/-/DC1
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Hox function in Drosophila MNs
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2038 RESEARCH ARTICLE