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First published online 2 January 2008
doi: 10.1242/dev.010876
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1 Department of Genetics, Cell Biology and Development, Howard Hughes Medical
Institute, University of Minnesota, 6-160 Jackson Hall, Minneapolis, MN 55455,
USA.
2 Institute of Neuroscience, Howard Hughes Medical Institute, University of
Oregon, Eugene, OR 97403, USA.
3 Department of Biological Sciences, University of Alberta, Alberta, T6G 2E9,
Canada.
4 Howard Hughes Medical Institute, University of Oregon, Eugene OR 97403,
USA.
5 Howard Hughes Medical Institute, University of Minnesota, 6-160 Jackson Hall,
Minneapolis, MN 55455, USA.
* Author for correspondence (e-mail: moconnor{at}umn.edu)
Accepted 29 October 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Activin, Brain, Drosophila, Larvae, Optic lobe, Proliferation
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). These
factors bind to a heteromeric receptor complex containing type I and II
transmembrane serine/threonine kinases. Upon ligand binding, the type I
receptor kinase is activated by the type II receptor and phosphorylates
members of the R-Smad family of transcription factors. TGF-β signaling
pathways are broadly divided into either the BMP or Activin/TGF-β
branches, based on which Smad transcription factors they phosphorylate
(Feng and Derynck, 2005). The
BMP family of ligands signal through R-Smads1/5/8 whereas the
Activin/TGF-β branch utilizes R-Smads2/3. Phosphorylation of R-Smads
facilitates their association with a common Co-Smad and retention of the
R-Smad-Co-Smad complex in the nucleus, where it regulates transcription of
target genes in association with a wide variety of co-factors
(Miyazono et al., 2006).
Drosophila employs both BMP and Activin-like signaling pathways to
regulate numerous developmental processes. Among the seven TGF-β type
ligands, Decapentaplegic (Dpp), Screw (Scw), and Glass bottom boat (Gbb) are
members of the BMP family, whereas Activin-β (Actβ) and Dawdle (Daw)
(Parker et al., 2004;
Serpe and O'Connor, 2006),
fall within the Activin/TGF-β branch. Two novel ligands, Maverick (Mav)
and Myoglianin, are sufficiently diverged to preclude clear assignment to a
particular ligand subfamily (Parker et
al., 2004). Both the BMP and Activin/TGF-β pathways employ
Punt as a common type II receptor, and pathway specificity is provided by the
type I receptors (Brummel et al.,
1999; Brummel et al.,
1994; Das et al.,
1999; Letsou et al.,
1995; Ruberte et al.,
1995; Serpe and O'Connor,
2006; Zheng et al.,
2003). Tkv and Sax bind BMP-type ligands and phosphorylate Mad,
whereas Babo binds Activin-like ligands and signals through Smad2 (also known
as Smox - FlyBase) (Das et al.,
1999; Serpe and O'Connor,
2006; Shimmi et al.,
2005a; Shimmi et al.,
2005b; Zheng et al.,
2003).
Parsing out the functional relationship between individual TGF-β
ligands and receptors and particular developmental processes is a difficult
process, especially in vertebrates, in which the numbers of ligands and
receptors are large. In general, this process involves biochemically matching
a particular ligand with one or more signaling receptors and comparing the
phenotypes produced by knockdown of the different signaling components. In
Drosophila, the contributions of BMP pathway components to numerous
developmental processes, including cell-fate specification, imaginal-disc
growth and patterning and synapse development, have been well studied
(reviewed by Parker et al.,
2004). By contrast, the Activin pathway is less well
characterized. Unlike mutations in the BMP pathway, mutations in Activin
signaling components have not been shown to directly affect differentiation
but instead appear to primarily regulate neuronal wiring and proliferation.
For example, clonal analysis of either babo or Smad2 mutants
has shown that this pathway regulates mushroom body remodeling during
metamorphosis (Zheng et al.,
2003), morphogenesis of ellipsoid body neurons in the adult
(Zheng et al., 2006) and motor
axon guidance in the embryo (Parker et
al., 2006; Serpe and O'Connor,
2006). Regarding proliferation, loss of both maternal and zygotic
babo leads to small brains and small but properly patterned wings
(Brummel et al., 1999),
indicating a potential role in regulating proliferation of larval mitotic
tissue.
Analysis of Drosophila Actβ and Daw ligand loss-of-function
phenotypes indicates that these two ligands probably regulate separate aspects
of neuronal wiring, as dominant-negative and RNAi constructs that reduce the
activity of actβ phenocopy the mushroom body remodeling defects
seen in babo and Smad2 mutants
(Zheng et al., 2003), whereas
null mutants of daw phenocopy the babo and Smad2
mutant motor axon guidance defects (Parker
et al., 2006; Serpe and
O'Connor, 2006). In neither case, however, was an obvious
proliferation defect reported for loss of either ligand.
In this paper, we investigate the role of the Drosophila Babo/Smad2 pathway in larval brain development. We show that mutations in babo and Smad2 result in small brains with altered innervation of photoreceptor axons within the lamina and medulla. In contrast to the wiring defects described above, however, we demonstrate that these aberrations are not caused by defects in photoreceptor innervation or changes in cell fate of target neurons within the brain. Instead, they result primarily from reduced proliferation within the optic lobe and central brain leading to a reduction in the size of the photoreceptor target field. We further demonstrate that the Babo receptor is required in neuroblasts and that the ligands Actβ and Daw function redundantly to control proliferation in the brain. These results suggest that while the two Drosophila Activin-like ligands have at least partially independent roles in regulating neuronal wiring, they have largely redundant roles in regulating proliferation within the brain.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), as has the strong Smad2mb388 allele
(Zheng et al., 2003).
Previously, we reported that babo32 homozygotes have very
low levels of BrdU incorporation in third-instar brains
(Brummel et al., 1999).
However, we have since found that the babo32 chromosome
contains a second hit, which when homozygous leads to developmental delay and
severely reduced cell proliferation (M.B.O., unpublished). Therefore, only
heterozygous combinations of babo alelles are used in these studies.
The dawex32 and dawex11 are strong and
moderate loss-of-function alleles, respectively
(Serpe and O'Connor, 2006),
whereas daw4 is a presumed null mutation
(Parker et al., 2006). The
Cyclin Ac8LR1 allele is a strong amorph
(Sigrist and Lehner, 1997) and
was obtained from the Bloomington Stock Center. For genetic rescue
experiments, babo mutant lines carrying various Gal4 drivers were
crossed to different babo mutant alleles carrying UAS transgenes. The
1407>Gal4 driver (Luo et al.,
1994) was obtained from K.-F. Fischbach and the
worniu>Gal4 has been previously described
(Albertson et al., 2004). The
da>Gal4, elav>Gal4, and ey>Gal4 lines were
obtained from the Bloomington Stock Center. Mutant babo alleles with
different Gal4 drivers and UAS transgenes were balanced by the
CyO::Tm6,Tb compound chromosome. Null babo
photoreceptor clones were generated by crossing
FRTG13babo52/CyO::Tm6,Tb flies to
FRTG13uasCD8GFP; eyGal4-uasFlp/CyO::Tm6,Tb flies.
GFP-positive MARCM clones were induced by heat shock at 36°C for 1 hour of
early second-instar larvae from the cross of elavGal4hsFlp;
FRTG13tubGal80/CyO and
FRTG13babow224UASmCD8GFP/CyO. To identify
daw, actβ double mutants, non-GFP, y larvae were
obtained from the stock daw/CyO, actin-GFP;
actβed80/Dp1:4 (y+, spa). All
crosses were done at 25°C.
The daw promoter-Gal4 line was generated by cloning a 9 kb PCR
fragment containing the first intron and upstream sequences using the
following primer pair 5'-CTGAGCCCCTACGTCTGTATGATATG-3' and
antisense 5'-GATCTTCTGGATCGCCTTTGGTTTCA-3' into the pPelicanGal4
plasmid, which is derived from pPelican
(Barolo et al., 2000).
Transgenic flies were generated by standard injection procedure.
Staging of larvae
Freshly hatched larvae were collected for 5 hours on apple-agar plates and
staged to white prepupal stages (120 hours for yw and 144 hours for
babo mutants).
Immunostaining
Larval brain lobes, together with imaginal discs, were dissected in PBS and
fixed in 3.7% formaldehyde in PBS for 1 hour at room temperature, and were
washed by PBS plus 0.1% Triton X-100 (PBT). Antibodies purchased from the
Developmental Studies Hybridoma Bank (DSHB) include mouse 24B10 (1/100
dilution), rat anti-Elav (7E8A10, 1/400), mouse anti-Dachshund (mAbdac2-3,
1/50), mouse anti-Robo 13C9, (1/50), mouse anti-Repo 8D12, (1/50 dilution),
mouse anti-BrdU G3G4 (1/100), anti-Arm 7A1 (1/500), mouse anti-Pros MR1A
(1/100) and mouse anti-Discs large (1/1000). Rabbit anti-phosphorylated
histone H3 (pSer10, H-0412) was from Sigma (1/400). Rabbit
anti-Cyclin A antibody was used at 1/500 dilution
(Whitfield et al., 1990;
Nakato et al., 2002). Rabbit
anti-Scrib was diluted at 1:2500 (Albertson
et al., 2004), and guinea-pig anti-Mira at 1:500
(Lee et al., 2006).
Anti-active Caspase-3 antibody was a gift from Idun Pharmaceuticals, and used
at 1/2000 dilution. Rat monoclonal N-Cadherin antibody, DUex8, was a gift from
L. Zipursky and was used at a 1/50 dilution. Fluorescent conjugated secondary
antibodies (Molecular Probes) were used at 1/200 dilution. All primary
antibodies were diluted in PBT and incubated with tissue samples at 4°C
overnight. Secondary antibodies were typically incubated with tissue samples
for 2 hours at room temperature. Confocal images were taken with a Zeiss Axio
confocal microscope or a Leica TCS SP2.
BrdU incorporation
Larval brain lobes and attached eye discs were dissected in PBS and
transferred to M3 complete medium containing 0.4 mg/ml BrdU (Roche) and
incubated at 25°C for 30 minutes before fixation. Fixed tissue was treated
in 2 N of HCl at room temperature for 30 minutes before the addition of
anti-BrdU monoclonal antibody.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Zheng et al., 2006). We
noticed that mutant third-instar larvae also showed variable defects in
photoreceptor innervation of the lamina and medulla
(Fig. 1A vs B). Differentiated
photoreceptor cells are normally organized as ommatidial units, each
consisting of eight photoreceptor cells, R1-R8, which send their axons through
the optic stalk into developing optic lobes. The axons of R1-R6 terminate and
form a neural plexus at the lamina, whereas the axons of R7 and R8 project to
the medulla (Clandinin and Zipursky,
2002) (Fig. 1A). In
babo mutant brains, photoreceptors R1-6 formed a lamina plexus that
was very reduced in size (Fig.
1B). In addition, the R7 and R8 axon projections to the medulla
were highly perturbed, and their growth cones formed bundles instead of a
regular lattice network typical of controls
(Fig. 1A vs 1B insets). This
defect was not corrected at later stages, as axon targeting was still highly
perturbed in 3-day-old babo mutant pupae, and rotation of the medulla
and lamina relative to one another did not take place
(Fig. 1D vs E). Smad2
mutants exhibited similar photoreceptor axon targeting defects, indicating
that the babo defect is caused by the loss of the canonical Activin
signaling pathway (Fig.
1C). To further characterize the babo phenotype, we also immunostained babo mutant brain lobes with anti-Dachshund (Dac) antibody to highlight lamina neuron precursor cells, and with anti-Elav to visualize differentiated neurons. In the most severe cases, babo mutants exhibited reduced numbers of lamina cap neurons (Fig. 2A vs B) and loss of Dac-positive cartridge neurons. In larvae with less severe phenotypes, the number of Dac-positive cells in each cartridge was not greatly different from that seen yw controls, but the total number of lamina cartridges in babo mutants is smaller than that in wild type (see Fig. S1C in the supplementary material).
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). In
addition, anti-N-Cadherin, which stains both photoreceptors and the medulla
neuropil (Lee et al., 2001),
also reveals a disruption in the medulla neuropil formation
(Fig. 2G,I vs H-J).
Glial cells have been implicated as intermediate targets for photoreceptor
axon targeting (Clandinin and Zipursky,
2002). Therefore, we examined babo and Smad2
mutants with the glia-specific Repo antibody and compared them to yw
controls. White prepupae of wild-type larvae showed three layers of
well-aligned glial cells at the lamina and evenly distributed glia at the
medulla cortex (Fig. 2E). By
contrast, babo and Smad2 mutants exhibited a reduced number
of glia, with some misalignment of the glial cells within both the lamina and
medulla (Fig. 1C and
Fig. 2F).
Although based on only a limited number of markers, our data suggest that differentiation of neurons and glia is not grossly disrupted in the optic lobe of babo and Smad2 mutants, raising the possibility that the observed retinal axon targeting defects may result from the production of fewer lamina and medulla progenitor cells within the optic lobe (see below).
Babo function is required in the optic lobe and not in photoreceptor axons
As Drosophila retinal axons have been shown to regulate
proliferation in the lamina via delivery of Hedgehog and the EGF-like ligand
Spitz to the target field (Huang and
Kunes, 1996; Huang and Kunes,
1998; Huang et al.,
1998), and because Drosophila Activin is expressed in
photoreceptors (see Fig. S1A in the supplementary material), we wished to
address whether babo function is required in photoreceptor axons or
the brain for proper photoreceptor axon targeting and optic lobe
development.
The babo gene is alternatively spliced, producing two protein
isoforms, Baboa and Babob, which differ in their
extracellular ligand-binding domain (Wrana
et al., 1994). The baboa isoform has been specifically
implicated in mushroom body remodeling
(Zheng et al., 2003), while
ectopic expression of either the Baboa or Babob isoform
rescues dorsal neuron morphogenesis defects of babo mutants. To
examine the isoform requirements for proper optic lobe development and axon
targeting, we expressed baboa and
babob individually or in combination using the ubiquitous
daughterless Gal4 driver (da>Gal4). Expression of the
baboa isoform alone rescued neither the photoreceptor axon
targeting nor brain size defects (Fig.
3B). By contrast, ubiquitous expression of
babob alone (Fig.
3A) or baboa and babob
together (data not shown) not only rescued photoreceptor axon targeting and
brain lobe defects, but also restored viability. These rescue data suggest
that the babob isoform alone, at least when overexpressed,
is sufficient to provide proper signaling activity for normal optic lobe
development, axon targeting and viability.
To address tissue- and cell-type-specific requirements for babo function, a number of different Gal4 drivers were employed. Using the eye-specific driver eyeless Gal4 (ey>Gal4), we found that expression of neither baboa, babob nor a combination of both in eye discs was able to rescue the babo mutant phenotype (Fig. 3C and data not shown). This suggested that babo function in the photoreceptors is not sufficient for directing normal photoreceptor axon targeting. We also used the FLP/FRT system to generate babo mutant clones in an otherwise babo heterozygous background. Large babo-mutant photoreceptor cell clones were found to project their axons normally into babo heterozygous brain lobes (Fig. 3E), providing further support for the argument that babo function in photoreceptor cells is dispensable for proper axon targeting and optic lobe development.
As glial cells provide both guidance cues and trophic factors for optic
lobe development (Hidalgo et al.,
2006), and because their numbers are reduced in babo
mutants, we asked if selective expression of babo in glial cells was
able to suppress any aspect of the babo mutant phenotype. As shown in
Fig. 3D, expression of
baboa and babob in glial cells using
the glia-specific driver repo>Gal4 did not alter the babo
mutant phenotype. By contrast, expression of baboa and
babob using the 1407>Gal4 driver
(Luo et al., 1994), which is
expressed in both neuroblasts and many differentiating neurons of the brain
but not in the eye disc (Fig.
3F), was able to rescue both brain size and the axon-targeting
defect (Fig. 4G). In addition,
the worniu>Gal4 driver that is expressed primarily in neuroblasts
(Albertson et al., 2004) and
ganglion mother cells (GMCs), but not the eye disc
(Fig. 3H and see Fig. S3 in the
supplementary material), was also able to rescue the babo mutant
optic lobe phenotype (Fig. 3I),
and some of these mutant animals survived to adults. Lastly, we found that
overexpression of the EcR-1B receptor, the only known target of
Actβ/Baboa signaling in the brain lobes, could not rescue the
axon-targeting defects (data not shown) in contrast to its ability to suppress
neuronal remodeling defects (Zheng et al.,
2003; Zheng et al.,
2006).
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).
However, it is not clear how proliferation is affected in babo mutant
brains. Because the optic lobes comprise the majority of brain lobe tissue,
and have been well characterized (Egger et
al., 2007), we focus on the growth and proliferation of the optic
lobe.
In wild-type white prepupae, optic lobes make up more than half of the
brain lobe. At late stages, the optic lobe epithelium produces many medulla
neuroblasts and laminar precursor cells (LPCs). These neuroepithelial cells of
the outer proliferation center (OPC) and inner proliferation center (IPC), as
well as medulla neuroblasts and LPCs, undergo rapid proliferation to produce
the cells of the lamina and medulla cortices
(Fig. 4B)
(Egger et al., 2007). As noted
above, in babo mutants the optic lobe is much smaller than in
wild-type controls. We find that the overall organization of the OPC and IPC
epithelia is normal and that they retain their ability to produce neuroblasts
and LPCs. However, there is approximately a 50% decrease in the number of
medulla neuroblasts (Fig. 4D),
a decrease in the number of ganglion mother cells produced from these medulla
neuroblasts, and a subsequent decrease in the number of Elav+
maturing neurons within the medulla cortex
(Fig. 4B,C). Furthermore, there
is a decrease in the number of LPCs and thus a decrease in the number of
lamina cartridges and laminar neurons (Fig.
2B and Fig. 4C, and
see Fig. S2B,C in the supplementary material). Similar results are observed
for central brain neuroblasts, where we observed a slight reduction in
neuroblast numbers (see Fig. S2A in the supplementary material). We observed
no evidence for an increase in apoptosis, as revealed by staining for
Caspase-3 (also known as Decay - FlyBase)
(Fig. 5N-P), confirming
previous results (Brummel et al.,
1999). We conclude that babo function is required for
generating the normal number of medulla neuroblasts and laminar precursor
cells in the optic lobe.
Neuroblasts proliferate more slowly in babo mutants
To further address whether proliferation rates are altered in babo
mutant optic lobes, we generated GFP-positive marked babo
loss-of-function clones using the MARCM system
(Lee and Luo, 2001). As there
are different patterns of mitotic clones within the developing optic lobes
(data not shown), we sought to compare only the babo mutant and
wild-type clones at similar positions within the optic lobe (example clones in
Fig. 4E vs F) and central brain
(Fig. 4H vs I). This analysis
revealed that babo mutant clones in the optic centers and central
brain contained 30-50% fewer cells than did wild-type control clones, which
agrees with the overall reduction of brain size and lamina cartridge size. We
conclude that babo mutants have reduced proliferation of optic lobe
and central brain neuroblasts.
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; Penton et al.,
1997).
We next examined the levels of different Cyclins in babo mutant
clones and fully mutant brains. We found no difference in the levels of Cyclin
B and E (data not shown); however, Cyclin A levels were enhanced in both
babo clones (Fig.
5J-L) and fully mutant brains
(Fig. 5H,I). Ectopic expression
of Cyclin A has been previously shown to accelerate the G1/S transition as
well as delay cells from exiting M phase
(Lehner and O'Farrell, 1989;
Lehner et al., 1991;
Sprenger et al., 1997). Thus,
elevated Cyclin A in the babo mutants could lead to the observed
decrease in M-phase cells. Consistent with this model, heterozygosity for the
Cyclin Ac8RL1 mutation substantially suppressed the
babo mutant phenotype (Fig.
5M). We conclude that loss of Babo/Smad2 signaling leads to
elevated Cyclin A levels, which contribute to the optic lobe proliferation
defects seen in babo mutants.
actβ and daw act redundantly to regulate optic lobe development
Previous work has demonstrated that both Drosophila Activin-β
and the Activin-like protein Dawdle signal through Activin type I receptor
Babo to Smad2 (Zheng et al.,
2003; Parker et al.,
2006; Serpe and O'Connor,
2006), suggesting that one or both are likely candidates for
regulating optic lobe development. In situ hybridization studies revealed that
both are expressed in the developing optic lobes
(Fig. 6A,B). In addition,
daw is also expressed in many glial cells, including surface glia
within the optic lobe, and Actβ is expressed in mushroom body neurons
(Fig. 6C)
(Serpe and O'Connor, 2006)
(and data not shown).
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We also examined several daw mutant lines for optic lobe morphology. We observed no photoreceptor axon-targeting defects in daw3/daw4 heteroallelic mutant larvae or the dawex11 homozygous larvae (data not shown). However, in a low percentage (4%, n=50) of dawex32 homozygous deletion mutants (Fig. 6D), we did observe photoreceptor axon targeting and optic lobe defects in white prepupae that were reminiscent of those seen in babo mutants (Fig. 6E). The low penetrance of the babo-like phenotypes in daw mutants, and the overlapping expression of actβ and daw in the optic lobes, suggested that these two genes might act redundantly to control growth in the optic lobes. To investigate this issue, we examined the phenotypes of various daw, actβ double mutant combinations. In the dawex11/dawex11, actβed80/actβed80 double mutants overall brain morphology was relatively normal. However, approximately 20% (n=30) of the mutant larvae exhibited collapsed and bundled R7 and 8 growth cones that were not seen in either single mutant (Fig. 6G). In the stronger dawex32/dawex32 actβed80/actβed80 allelic combination, the penetrance of the severe small brain and axon-targeting phenotype seen in dawex32 mutants alone was increased from 4 to 50% (n=30). In the daw4/dawex32, actβed80/actβed80 combination, larvae died during late second- or early third-instar stages, precluding us from examining the brain phenotype. We conclude that Actβ and Daw function redundantly to control proliferation in the optic centers of the Drosophila larval brain.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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It is not entirely clear how Activin signaling in neuroblast lineages
maintains the wild-type number of brain neuroblasts. Approximately 100
neuroblasts per brain lobe are formed during embryogenesis, and most go
quiescent at the embryonic/larval transition. From L1-L3 they progressively
re-enter the cell cycle to resume their cell lineages. Perhaps the slower cell
cycle of Activin pathway mutants inhibits exit from quiescence and promotes
premature differentiation. Mutations in trol, which encodes a heparin
sulfate proteoglycan Perlecan, prevent neuroblast reactivation and lead to a
severe reduction in neuroblast numbers and brain size
(Datta, 1995;
Park et al., 2003). Many
TGF-β ligands bind to heparin sulfate proteoglycans, and thus part of the
effect of Activin signaling on brain size might be mediated by Perlecan or
other proteoglycans such as the glyipican Dally (see below).
The small brain size is not just caused by a reduced number of neuroblasts,
however; babo mutant clones that contained a single neuroblast
produced fewer daughter cells in a given time window than did wild-type
neuroblasts, presumably due to the increased expression of Cyclin A and the
delay in metaphase exit. One additional possibility is that the Activin signal
may affect neuroblast temporal identity progression in larval neuroblast
lineages, similar to the effect of temporal identity mutations on embryonic
neuroblast lineages (Isshiki et al.,
2001), leading to the failure to produce early, mid or late
subsets of larval lineages. Testing this hypothesis awaits the development of
markers for different neurons within optic lobe neuroblast lineages.
The importance of Activin in regulating neuroblast proliferation is
reminiscent of the positive role that Activin/Nodal signaling plays in
regulating the cell cycle of mouse and human ES cells
(James et al., 2005;
Ogawa et al., 2006). In those
cells, as in Drosophila neuroblasts, Activin/Nodal signaling
enhances, but is not absolutely required for, cell proliferation
(Ogawa et al., 2006). Another
point of potential similarity is that the mES cells endogenously produce an
Activin/Nodal signal leading to an autocrine/paracrine regulation of
proliferation. While our in situ data are not of sufficient resolution to
unambiguously assign expression of actβ and daw to
particular cell types, both are expressed in the optic proliferation zones
where neuroblasts are highly concentrated. It is also possible that some
ligand may be supplied by the innervating photoreceptors. Activin is strongly
expressed in R7 and 8 (Ting et al.,
2007) and like hh and spitz may provide a tropic
signal that simulates proliferation in the target tissue
(Huang and Kunes, 1996;
Huang et al., 1998).
Lastly, it is interesting to note that Activins are not the only
TGF-β-like factors required for proliferation of Drosophila
neuroblasts. The BMP family member Dpp is expressed in four regions in each
brain lobe (Kaphingst and Kunes,
1994; Yoshida et al.,
2005). Two lie in the dorsal and ventral margins of the posterior
optic zone neuroepithelium near what has been termed the lamina glial
precursor region (Yoshida et al.,
2005), whereas the other two smaller zones are more interior at
the base of the inner proliferation zone. In the brain, the dpp
loss-of-function phenotype is remarkably similar to that seen in babo
mutants (Kaphingst and Kunes,
1994; Yoshida et al.,
2005). A potential trivial explanation for the similarity in
phenotypes might be that Activin signaling is required for dpp
expression, or vice versa. However, dpp is still expressed in
babo mutants (see Fig. S1C,D in the supplementary material) and
daw and actβ are both still expressed in dpp
mutants, although it is difficult to know in each case whether the levels are
equivalent. Thus, both Dpp and Activin signaling appear to be required to
stimulate brain neuroblast proliferation.
In addition to regulating proliferation in the brain, Dpp signaling plays a
major role in regulating proliferation in other tissues, including the
imaginal discs (Burke and Basler,
1996; Rogulja and Irvine,
2005). Once again, Activins may collaborate with BMPs in
regulating proliferation in this tissue. In particular, we note that
babo mutants show ectopic P-H3 staining within the morphogenetic
furrow of the eye disc, which is also observed in loss-of-function mutants in
the Dpp receptor Tkv (Horsfield et al.,
1998). Furthermore, babo mutant wing disc clones can grow
large in contrast to clones mutant in dpp signaling components
(Burke and Basler, 1996),
although the overall sizes of babo mutant discs are not affected
proportionally as much as is the brain (C.C.Z. and M.B.O., unpublished).
Therefore, the way in which BMP and Activin inputs regulate the cell cycle
might be different in discs versus the brain, or the two tissues might exhibit
different sensitivities to common inputs.
How Activins and BMPs affect the cell cycle is not entirely clear. In the
wing disc, Dpp signaling through Tkv/Mad has been shown to promote the G1-S
transition (Martin-Castellanos and Edgar,
2002). In the brain, babo mutants exhibit a decrease in
the M/S ratio, which could be due to a decrease in cells at the G2/M phase of
the cell cycle. Consistent with this view, we find that Cyclin A levels are
enhanced in babo mutants and that heterozygosity for a Cyclin
A mutation suppresses the babo phenotype. This is very similar
to that seen in dally mutants, which also affect brain development by
causing a delay in the G2-M transition within the outer proliferation centers.
Just as we have found for babo mutants, heterozygosity for Cyclin
A suppresses the dally cell cycle defect
(Nakato et al., 2002).
Given the results described above, one attractive model for how both BMPs
and Activins contribute to cell cycle progression is that they regulate the
cycle at different points: Activins at G2-M and BMPs at G1-S. Alternatively,
as previous work has suggested that Cyclin A probably has roles in regulating
both G2-M and G1-S transitions in Drosophila
(Lehner and O'Farrell, 1989;
Lehner et al., 1991;
Sprenger et al., 1997) and as
Smads can form heterotrimers (Chacko et
al., 2004), it may be that a composite signal composed of a
Smad2/Mad/Medea heterotrimer acts at several points in the cell cycle.
Interestingly, several potential target genes that are regulated by both
Activin and BMP signals in the larval brain have been identified by microarray
studies using activated receptors (Yang et
al., 2004), but no obvious candidates for genes that might
influence proliferation are evident within the list.
Lastly, we note that daw has been demonstrated previously to play
a role in motoneuron axon guidance in the embryo, while actβ has
been implicated in mushroom body remodeling
(Zheng et al., 2003) and more
recently in regulating the terminal steps in photoreceptor R8 targeting during
pupal stages (Ting et al.,
2007). As both ligands are expressed in each of these tissues
(P.A.J. and M.B.O., unpublished), it is possible that there may be functional
redundancy that limits the severity of the previously observed phenotypes.
Consistent with this view, we have recently found that both actβ
and daw modulate neurotransmission at the neuromuscular junction and
that the double mutant phenotypes are more severe than those seen in the
single mutants (Yi Ren and M.B.O., unpublished), similar to their redundant
function in regulating neuroblast proliferation described here. In conclusion,
all available data suggest that Activin signaling plays at least two important
roles in Drosophila nervous system development. First, it ensures
that the proper numbers of cells are produced in the CNS; and second, it helps
establish correct functional connections between neurons and their synaptic
partners.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/3/513/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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5%) of daw32 homozygotes show optic lobe
and axon targeting defects reminiscent of babo mutants. (F) Optic lobes
developed normally and photoreceptor axons target correctly in
actβed80 mutant larvae. (G) Double
mutants of the genotype
dawex11:actβed80 exhibit altered
R7 and R8 growth cone bundling and morphology. (H) The
dawex32;actβed80 double
mutants showed significantly enhanced penetrance of the strong optic lobe
phenotype. Central bl, central brain lobe.