|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online November 21, 2006
doi: 10.1242/10.1242/dev.02673
1 Department of Developmental and Cell Biology, University of California Irvine,
Irvine, CA 92697-2300, USA.
2 NIH/NIDCR Bethesda, MD 20892, USA.
3 Developmental Biology Center, University of California Irvine, Irvine, CA
92697-2300, USA.
* Author for correspondence (e-mail: karora{at}uci.edu)
Accepted 3 October 2006
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Drosophila, TGF-ß, Alp23B, Activin, Baboon, Smad2 (Smox), Motoneuron, Axon pathfinding, Neuromuscular system, Glia
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Dickson,
2002; Tessier-Lavigne and
Goodman, 1996).
In Drosophila, embryonic motoneurons contact their synaptic
targets in the bodywall musculature in a highly stereotypic manner, providing
a powerful system for genetic analysis
(Bate and Broadie, 1995;
Keshishian et al., 1996).
Extensive screens have identified additional molecules that control axon
guidance and target recognition, such as the IgCAM Fasciclin 2 (Fas2) that
promotes axon-axon adhesion and fasciculation
(Lin and Goodman, 1994), as
well as Beaten path (Beat-Ia) and Sidestep (Side) that trigger defasciculation
at specific choice points (de Jong et al.,
2005; Fambrough and Goodman,
1996; Pipes et al.,
2001; Sink et al.,
2001; Van Vactor et al.,
1993). The receptor protein tyrosine phosphatase (RPTP) Lar and
its homologs constitute another class of molecules that affect axon guidance,
a role that appears to be conserved in C. elegans and mice
(Desai et al., 1996;
Desai et al., 1997;
Krueger et al., 1996;
Schindelholz et al., 2001;
Sun et al., 2001). Syndecan
and Dally-like function as ligands for Lar, implicating heparan sulphate
proteoglycans (HSPGs) in axon pathfinding as well
(Fox and Zinn, 2005;
Johnson et al., 2006). The
pathways that link the activity of guidance cues to changes in cytoskeletal
dynamics are beginning to be resolved through the demonstration that proteins
such as Rho GTPases, the GEF Trio and Ena/VASP, also affect axon extension
(Huber et al., 2003;
Luo, 2002). Cytoskeletal
components that alter growth cone motility include Short stop (Shot), Profilin
and Drosophila Pod1 that regulate actin and microtubule dynamics
(Lee et al., 2000;
Rothenberg et al., 2003;
Wills et al., 1999).
Interestingly, genetic screens have identified few regulators of guidance
factors and signaling protein expression, although they must play an equally
important role in enabling pathfinding.
In this paper, we provide evidence that Dawdle (Daw), a Transforming Growth
Factor-ß (TGF-ß) superfamily ligand, acts through an activin
signaling pathway and controls motoneuron pathfinding during
Drosophila embryogenesis. Activins, along with the prototypical
TGF-ßs and Bone Morphogenetic Proteins (BMPs), constitute three
structurally and functionally distinct subfamilies of the TGF-ß
superfamily (Massague, 1998).
TGF-ß family members have multiple functions in the nervous system, such
as neural induction and patterning, and regulation of neuronal survival,
differentiation and synaptogenesis (Altmann
and Brivanlou, 2001; Mehler et
al., 1997; Stern,
2005; Unsicker and
Krieglstein, 2002). However, a role for activin signaling in
neuronal pathfinding has not been demonstrated previously in any system.
Activins, like other TGF-ß ligands, signal by binding a heteromeric
receptor complex of type-I and type-II transmembrane serine-threonine kinases.
Ligand binding results in type-I receptor activation and consequent
phosphorylation of a receptor-specific member of the Smad family of
signal-transduction proteins, which then associates with a co-Smad and
translocates into the nucleus to directly regulate gene expression
(Massague, 1998;
Shi and Massague, 2003). Both
Drosophila type-II receptors, Punt (Put) and Wishful Thinking (Wit),
can bind BMPs and activins; thus, signaling specificity is dependent on
recruitment of the appropriate type-I receptor to the complex
(Childs et al., 1993;
Letsou et al., 1995;
Marques et al., 2002;
Ruberte et al., 1995). The
activin pathway in flies is represented by a single type-I receptor Baboon
(Babo) and the signal-transducer Smad2 (Smox - Flybase), the fly homolog of
activin-specific Smad2/3 (Brummel et al.,
1999; Das et al.,
1999; Wrana et al.,
1994). The Drosophila genome contains four potential
ligands for Babo: Activin, Myoglianin (Myg), Maverick (Mav) and Daw (reviewed
by Parker et al., 2004). Both
Activin and Myg bind Babo in biochemical assays, but only Activin has been
shown to stimulate Smad2 phosphorylation
(Lee-Hoeflich et al., 2005;
Zheng et al., 2003). The
absence of mutations in these four ligands has made it difficult to establish
their developmental roles. However babo and Smad2 mutants
display phenotypes such as reduced cell proliferation in the larval brain and
defects in ecdysteroid-dependent remodeling of mushroom body neurons,
implicating activin signaling in neuronal development
(Brummel et al., 1999;
Zheng et al., 2003). Important
questions that remain to be addressed are the identities of ligands that
trigger babo activity in vivo and the contributions they make.
Here we present biochemical and genetic evidence that Daw initiates an activin signaling pathway via the receptors Put and Babo, and the transducer Smad2. Mutations in daw display premature stalling and defasciculation defects in embryonic motoneuron pathfinding. Interestingly, although Daw is expressed in the glia and muscles, the receptor Babo appears to be required in the motoneurons. Furthermore, our data suggest that Daw plays a permissive rather than an instructive role, potentially by enabling the response of the growth cone to other spatial cues. These results provide the first demonstration in any system that a canonical activin signaling pathway is involved in axon guidance.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
I (isoforms a and b),
UAS-tkvI (also known as
TkvGSK), UAS-putI and
OK6-Gal4 (M. B. O'Connor, HHMI, University of Minnesota, MN), dMef2-Gal4 (E.
Olson, University of Texas Southwestern Medical Center, Dallas, TX),
Repo-Gal4 (V. Auld, University of British Columbia, Vancouver,
Canada). Maternal/zygotic babo-null embryos were derived from
germline clones induced in FRTG13, babo52/FRTG13,
ovoD females crossed to babo32/CyO wg-lacZ
males.
Molecular and genetic characterization of daw
pCS2+Daw contains an SspI-StuI fragment from GH14433;
pUAS-Daw contains a 2.5 kb EcoRI-NotI fragment from
pCS2+Daw. Daw-XmnI contains a 7.8 kb fragment from P1 clone DS07149 in
pCaSper. daw3 and daw4 were generated
in a b, pr, cn, wx, bw background. Deletion end-points and lesions
were determined by sequencing PCR-amplified DNA from homozygous daw
mutant embryos. The isogenized parental stock and both EMS alleles contain two
polymorphisms as compared with the Drosophila genome sequence: a
His143
Tyr substitution and an in-frame 9 bp deletion corresponding to
Ser173-Pro174-Leu175. The parental stock is viable and does not show
pathfinding defects, indicating that these substitutions do not have
functional consequences.
Lethal phase analysis
Egglays from daw-/CyO, Kr-Gal4, UAS-GFP stocks were
collected on agar plates at 25°C in 70% humidity. Hatched larvae were
counted and homozygous mutants transferred to a fresh plate. Dead larvae were
staged by mouth hook morphology. Pupae were left on plates and hatching adults
transferred to fresh vials. Data represent averages from 3 trials
(n=600).
Immunohistochemistry and in situ hybridization
DIG-labeled daw and babo riboprobes were used for in situ
hybridization (Tautz and Pfeifle,
1989). For histochemistry, embryos were stained with 1D4 (1:100),
BP102 (1:200) or 
-Repo (1:200) antibody (Developmental Studies
Hybridoma Center) and -mouse biotin-conjugated secondary antibody, and
visualized using the Vectorstain ABC Kit. daw- and
babo- embryos were distinguished by the absence of
CyO, wg-lacZ marker. Smad2 and put mutants were
identified by the absence of Twist-GFP. Embryos were analyzed using
Nomarski optics.
Signaling assays
Cell signaling assays were carried out in S2 cells as previously described
(Zheng et al., 2003).
Smad2-FLAG, Babo and BaboI cDNAs were cloned into pPac. Daw, Put and
PutI cDNAs were cloned into the inducible vector pRmHA-1Mt.
BaboI and PutI were obtained from M. B. O'Connor. Proteins were
separated on 12% SDS-PAGE and transferred to nitrocellulose. Phosphorylated
and total Smad2-FLAG were detected using rabbit -PS2
(Faure et al., 2000) and mouse
-FLAG (Sigma), respectively, and the ECL Plus Kit (Amersham).
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
) henceforth refer to the locus as daw. The
daw ORF shows several features characteristic of TGF-ß
superfamily ligands (Fig. 1B).
The first in-frame methionine is followed by a signal sequence indicating that
the protein is likely to be secreted. Two consensus cleavage sites for
furin-like proteases, RRRK (464-467) and RQKR (469-472), would generate a
mature ligand of 114 (or 119) amino acids. Within the ligand domain, Daw
shares 35% identity with human activinC, TGF-ß3 and BMP3
(Fig. 1C). However, the
presence of nine rather than seven cysteines at stereotypic positions
characteristic of activin and TGF-ß subfamilies, suggests that Daw
represents a divergent activin/TGF-ß ligand in Drosophila
(Fig. 1C).
Analysis of daw expression revealed maternally-provided mRNA in
presyncitial stage embryos (Fig.
2A). Zygotic daw transcripts were seen in the mesoderm
from stage 6 to 8, and at higher levels after stage 9
(Fig. 2B,C). At stage 13,
expression was also detected in the visceral mesoderm and oenocytes
(Fig. 2D,E). Somatic muscle
expression is significantly reduced by stage 15. Instead, at stage 16,
prominent expression was seen in the ventral nerve cord (VNC), in median,
intermediate and lateral groups of cells in a segmental pattern
(Fig. 2F,G,I,I'). Absence
of daw expression in the VNC of glial cells missing
(gcmN7-4) mutants that lack glia
(Fig. 2J)
(Jones et al., 1995), as well
as double staining with -Repo (data not shown), indicated that the
daw-positive cells in the VNC correspond to glia. Additional sites of
transcription were the fat body (Fig.
2G), the ring gland, cells in the maxillary segment, hindgut and
the posterior spiracles (Fig.
2H). In larvae, daw was expressed in the outer
proliferative center of the optic lobe and in the central brain
(Fig. 2K), the wing and leg
imaginal discs (Fig. 2L,M), and
in larval bodywall muscles (Fig.
2N). Northern blots detected a 2.7 kb transcript at high levels in
late embryonic and larval stages and in adult males and females (data not
shown).
|
1 and
daw2 that remove 2.1 kb and 1.4
kb of DNA, respectively (Fig.
1A). Both deletions eliminate the first exon of daw-A and
the translation start site in the second exon. In a screen for lethal loci
uncovered by Df(2L)C144
(Littleton and Bellen, 1994),
we isolated two EMS alleles daw3 and
daw4 that fail to complement
daw1. daw3 contains
a 2 bp deletion at 1893-1894 (Fig.
1B), leading to a frame shift in the prodomain and production of
66 altered residues before termination. A GT change at position 1819 in
daw4 introduces a premature termination codon, also in the
prodomain. In both cases, the resulting mutant proteins (Met1-Leu515 and
Met1-His425, respectively) would lack the ligand domain. The phenotype of
daw3 and daw4 in trans to
Df(2L)C144 is indistinguishable from homozygous mutants, suggesting
that they represent strong loss-of-function or null alleles. A 7.8 kb genomic
rescue construct that includes both daw isoforms (Daw-XmnI,
Fig. 1A) rescues all four
alleles, indicating that the observed lethality results from loss of
daw activity.
|
|
-Fas2) revealed
occasional breaks and fusions in the longitudinal axon fascicles at a low
incidence (5%) (Fig. 3A,B).
Examination of the glial cell population in daw mutants with
-Repo antibodies showed no overt defects
(Fig. 3C). In addition,
neuronal cell fate markers including even skipped, engrailed, achaete,
eagle, Fasciclin 3 and gooseberry were unaltered in mutant
embryos, suggesting that daw is not involved in cell fate
specification (data not shown).
We next analyzed motoneuron guidance in daw- embryos
stained with 1D4. During late embryogenesis, approximately 40 motoneurons in
each abdominal hemisegment innervate bodywall muscles in a stereotypic fashion
(reviewed by Bate and Broadie,
1995; Keshishian et al.,
1996; Landgraf et al.,
1997). Motoneurons exit the VNC via two routes, the segmental
nerve (SN) and the intersegmental nerve (ISN), and project into the muscle
field through five major pathways: the main ISN, ISNb, ISNd, SNa and SNc
(Fig. 3D). In
daw- embryos, ISNb and SNa axons exited the VNC correctly
and extended into their target field, but failed to advance completely and
innervate the appropriate muscles (Fig.
3E). In wildtype embryos, ISNb fasciculates with the ISN for a
short distance, before it enters the ventral muscle domain and innervates the
ventral longitudinal muscles (VLMs). Axons in ISNb innervate muscles 6/7, or
extend dorsally to muscle 13, while a subset bypass muscle 13 to innervate
muscle 12 (Fig. 3D,F) (for
details, see Landgraf et al.,
1997). Analysis of ISNb defects in different daw alleles
showed that in rare instances (1-6% hemisegments) the axons stalled at muscle
6/7 and failed to form synapses on muscles 13 and 12. More commonly, ISNb
axons terminated at muscle 13 (11-21%, open arrowhead in
Fig. 3G), or reached muscle 12
but were unable to form a synapse (6-17%, arrow in
Fig. 3G, data for individual
alleles in Fig. 3J). In total,
23-38% hemisegments in daw- embryos displayed some defect
in ISNb pathfinding.
Similar defects were seen in the context of SNa pathfinding. In wild type, the SNa extends dorsally past the VLMs to the lateral muscles, where it bifurcates to form a dorsal branch that innervates muscles 21-24, and a lateral branch that innervates muscles 5 and 8 (Fig. 3D,H). In daw- embryos, SNa extended into the lateral muscle field correctly but frequently exhibited loss of one or both branches (12-21% of hemisegments, Fig. 3E, arrow in Fig. 3I). In a few instances, the SNa stopped short of the bifurcation choice point (2-4%, Fig. 3K). In both the ISNb and SNa pathways, we frequently observed a thickening at or in the vicinity of the affected branchpoints, suggesting that axons stalled prematurely (open and closed arrowheads in Fig. 3G,I). We also observed minor defects in the ISN, but the ISNd and SNc trajectories appeared unaltered (data not shown).
|
2/daw2
males, the range of axon guidance defects was qualitatively similar to zygotic
nulls, but the incidence increased to 50% ISNb and 27% SNa defects
(Fig. 3J,K), indicating that
maternally-provided Daw contributes to neuronal pathfinding. By comparison,
wild-type embryos displayed only 4% defects in ISNb and 2% in SNa
(Fig. 3J,K). Taken together,
these results indicate that daw is required for axon guidance or
outgrowth of ISNb and SNa axons.
Daw signals via the activin type-I receptor Babo and Smad2
The presence of nine conserved cysteines in the Daw ligand domain suggests
that it belongs to the activin/TGF-ß subfamily. In Drosophila,
activin signaling is mediated by the type-I receptor Babo, which can associate
with either Put or Wit type-II receptors
(Brummel et al., 1999;
Lee-Hoeflich et al., 2005;
Wrana et al., 1994). In both
cases, complex formation results in phosphorylation of Smad2 but not of the
BMP-specific Smad Mothers against dpp (Mad). Furthermore, both babo
and Smad2 are involved in axon remodeling in the larval mushroom
body, linking their activities in vivo
(Zheng et al., 2003).
wit null embryos show no axon guidance defects
(Marques et al., 2002), making
Put a more likely type-II receptor for Daw. To establish the Daw signaling
pathway, we assayed its ability to mediate Smad2 phosphorylation in
transiently transfected Drosophila S2 cells
(Fig. 4A). Cells transfected
with Smad2 alone showed no basal phosphorylation when probed with -PS2
antisera (Faure et al., 2000)
that recognizes phosphorylated Smad2. However, low levels of activation could
be seen in the presence of Babo, without addition of ligand
(Fig. 4A, lanes 1, 2). In cells
challenged with Daw-conditioned media, we observed a significant increase in
Smad2 phosphorylation that was further enhanced upon cotransfection with Babo
(lanes 4, 5). The response of S2 cells to ligand alone suggests that
endogenous receptor levels are sufficient to allow Daw signaling to occur (see
lane 2), consistent with the finding that S2 cells express babo and
put
(http://flight.licr.org/).
To resolve the identity of the endogenous receptor, we cotransfected a
dominant-negative form of Babo (BaboI) that retains the ligand-binding
domain but lacks the intracellular kinase domain required for signaling.
Similar dominantnegative Tkv and Sax receptors are known to disrupt BMP
signaling in a ligand-specific manner
(Haerry et al., 1998;
Neul and Ferguson, 1998;
Nguyen et al., 1998).
Expression of BaboI blocked the response to Daw
(Fig. 4A, compare lanes 4, 6),
suggesting that the endogenous Daw signaling complex includes Babo.
|
I (Fig.
4B, compare lanes 10, 12). Thus, our data demonstrate that Daw
signal-transduction requires the activin pathway components Put and Babo, and
triggers phosphorylation of Smad2.
Activin pathway mutants show similar axon guidance defects to daw mutants
To determine whether Daw utilizes a canonical signaling pathway to regulate
growth cone guidance, we assayed whether mutations in put, babo and
Smad2 disrupt motoneuron pathfinding. Both put and
Smad2 are enriched in the embryonic mesoderm and nervous system
(Brummel et al., 1999;
Childs et al., 1993), making
them plausible candidates for mediating Daw signaling in vivo. We found that
babo transcripts were ubiquitously distributed and highly enriched in
the brain and the VNC during stages 13-17
(Fig. 5A,B). daw, babo
and Smad2 expression also overlapped at later stages; for example, in
the optic lobe, leg and wing discs (Fig.
2) (Brummel et al.,
1999; Das et al.,
1999).
Analysis of motoneuron pathfinding in babo- embryos revealed defects similar to those observed in daw mutants. In zygotic null babo32 animals, both ISNb and SNa branches entered their muscle fields correctly but failed to extend correctly. In 24% of hemisegments, ISNb axons stalled prematurely (Fig. 5C,D), and in 20% of hemisegments the SNa failed to defasciculate resulting in loss of either dorsal or lateral branches (Fig. 5G, arrows). Both ISNb and SNa pathways showed a thickening of the nerve in the vicinity of the disruption, consistent with a stalling defect (Fig. 5G, arrowhead). We generated babo germline clones, and found that the penetrance of defects increased to 58% in ISNb and 31% in SNa pathfinding (Fig. 5C).
Determining the contribution of the type-II receptor Put to motoneuron
pathfinding is complicated by its requirement for dorsal cell fate
specification during early embryogenesis in response to the BMP ligands
Decapentaplegic and Screw (Letsou et al.,
1995; Ruberte et al.,
1995). Consequently, embryos lacking put function are
ventralized. To circumvent this problem, temperature-sensitive
put88 embryos (Simin
et al., 1998) were raised at the permissive temperature (18°C)
until stage 14, then shifted to restrictive temperature (25°C). These
embryos displayed no overt patterning defects, but did show motor axon
guidance defects. ISNb axons were stalled in 31% of hemisegments
(Fig. 5C,E), and 32% of SNa
axons failed to branch or extend completely
(Fig. 5C,H). Whereas the
penetrance of SNa pathfinding defects was comparable to daw or
babo nulls, the lower incidence of ISNb defects may reflect the fact
that our assay conditions do not completely eliminate put
function.
Analysis of mutations in Smad2 also revealed pathfinding defects
reminiscent of zygotic loss of daw and babo. In
Smad2388 mutants, 21% of hemisegments had ISNb defects and
7% showed loss of lateral or dorsal SNa branches
(Fig. 5C,F,I). The milder
phenotype, as compared with babo and daw nulls, is likely to
reflect perdurance of maternal product, as maternal/zygotic
Smad2-null embryos have a phenotype equivalent to loss of ligand or
type-I receptor (Serpe and O'Connor,
2006).
To further establish that Daw and components of the activin signaling pathway regulate axon guidance, we examined genetic interactions between daw, babo and put. In put88/+ and babo32/+ single heterozygotes, less than 4% of hemisegments showed abnormal ISNb pathfinding. In each case, heterozygosity for daw further enhanced the phenotype to 14% and 20%, respectively (Table 2), which is equivalent to zygotic loss of Daw signaling. We conclude that Put, Babo and Smad2, three components necessary for Daw signal-transduction in biochemical assays, are also required for ISNb and SNa axons to extend and form correct synapses. Furthermore, the close correlation of the phenotypic defects encountered in daw mutants and components of its signaling pathway, as well as the genetic interactions observed between these genes, suggest that the effect of Daw on axon guidance is mediated through a canonical activin signaling pathway.
|
|
|
I is expressed
in muscles (dMef2-Gal4), glia (Repo-Gal4), neurons (Elav-Gal4), or
specifically in motoneurons (OK6-Gal4), from stages 13, 12, 12 and 15,
respectively (Aberle et al.,
2002; Marques et al.,
2002; Ranganayakulu et al.,
1996; Sepp et al.,
2001). We found that only expression in motoneurons, but not in
muscles or glia, was effective in disrupting ISNb and SNa pathfinding. Embryos
expressing a single copy of UAS-baboI in motoneurons
showed 35% ISNb pathfinding defects and 5% SNa branching defects
(Fig. 6A,
Table 3). Using four copies of
UAS-baboI increased the incidence of SNa pathfinding
defects to 22% (Table 3),
suggesting that the ISNb and SNa pathways are differentially sensitive to Daw
signaling, or express different levels of Babo protein. Essentially similar
results were obtained when four copies of UAS-baboI
were expressed in post-mitotic neurons using Elav-Gal4
(Table 3). Dominant-negative
receptors corresponding to babo-a and babo-b isoforms, which encode different
extracellular domains and potentially display altered ligand
specificity/affinity (Wrana et al.,
1994), gave comparable results suggesting that Daw binds to both
receptor subtypes. By contrast, expression of
UAS-baboI in muscles resulted in just 7% of
hemisegments showing the weakest class of ISNb defects (loss of synapse on
muscle 12), and no defects in SNa branching. When Babo function was disrupted
in glia, only 4% of hemisegments showed ISNb defects and 2% displayed loss of
one SNa branch. Thus, loss of Daw signaling in glia and muscles resulted in
defects comparable to wild-type and control UAS-baboI
embryos (Table 3). A
neuron-specific requirement for reception of Daw signal was further reinforced
in experiments using the dominant-negative Put receptor. In animals carrying
UAS-putI and OK6-Gal4, 33% of hemisegments showed
disruption of ISNb pathfinding (Fig.
6B, Table 3),
recapitulating the defects encountered in daw, put and babo
mutants. We observed only a 3% incidence of pathfinding errors in SNa axons,
reflecting their reduced sensitivity. Taken together, these results suggest
that Babo and Put function is required in the motoneurons.
|
I and PutI, we assayed whether a dominantnegative
BMP-receptor TkvI had similar consequences. Expression of a single copy
of UAS-tkvI in the wing disc disrupts wing venation
and growth, consistent with loss of BMP signaling
(Haerry et al., 1998). By
contrast, expression of UAS-tkvI in motoneurons did
not result in significant pathfinding defects (4% in ISNb, 1% in SNa,
Table 3). Increasing the copy
number of UAS-tkvI did not increase the penetrance,
suggesting that the phenotypes caused by expression of BaboI and
PutI are specific and represent disruption of an endogenous activin
signaling pathway in motoneurons.
Restricted transcription of daw is not important for its function
In order to determine which site of Daw transcription (muscle or glia) is
important for axon guidance, we assayed the ability of tissuerestricted Daw
expression to rescue ISNb defects in daw- embryos. We
found that driving one copy of UAS-daw in muscles using dMef2-Gal4,
or in glia with Repo-Gal4, decreased the incidence of ISNb defects in
daw1 homozygous embryos,
whereas two copies of UAS-daw provided almost complete rescue (89%
and 95%, respectively), and reduced the incidence of defects to wild-type
levels (Table 4). Given that
daw encodes a secreted ligand, these data suggest that restricted
transcription of daw is not crucial for its function. Ectopic
expression of a single copy of UAS-daw in motoneurons also reversed
the defects encountered in mutant embryos
(Table 4). Interestingly,
excess or ectopic Daw did not perturb motoneuron pathfinding in any way. These
results support a model in which daw provides a permissive signal
whose effect is restricted by other guidance cues. Alternatively, its function
could be restricted to a subset of motoneurons through the action of other
molecules that regulate Daw activity.
|
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
An activin signaling pathway is involved in axon guidance in Drosophila
Cell signaling assays and phenotypic analyses indicate that Daw affects
motoneuron pathfinding by acting through Put, Babo and Smad2. Supporting this
idea, the incidence of ISNb pathfinding defects increases when animals with a
single copy of the receptors Put and Babo are further depleted of Daw ligand
(Table 2). Mutations in Daw and
its receptors result in a similar range and penetrance of phenotypes, arguing
that Daw is the primary contributor to activin signaling in motoneuron
pathfinding and that the canonical pathway can fully account for the ability
of Daw to influence axon guidance. The slightly higher penetrance of ISNb
defects in babo as compared with daw maternal/zygotic nulls
(59% versus 50%, Fig. 5C),
raises the possibility that an additional ligand could contribute to embryonic
motor axon guidance. Both Activin and Myg can bind Babo
(Lee-Hoeflich et al., 2005;
Zheng et al., 2003), and are
expressed in neural or muscle cells compatible with such a role
(Lo and Frasch, 1999).
Intriguingly, overexpression of Activin (and to a lesser extent Myg) can
partially rescue daw- pathfinding defects (L.P. and K.A.,
unpublished). However, an assessment of their roles in axon pathfinding must
await the recovery of mutations in these genes. Furthermore, daw may
have other functions in addition to embryonic pathfinding. A majority of
daw mutants die during pupal stages despite the fact that pathfinding
defects are largely corrected by the third larval instar (L.P. and K.A.,
unpublished).
Daw is likely to function by signaling to motoneurons
Daw could act as a paracrine signal from the muscle or glia to influence
motoneurons. Alternatively, it could provide an autocrine signal that supports
glial or muscle growth/function and affects axon outgrowth indirectly. Our
data show that cell-autonomous disruption of activin signaling in muscles or
glia does not disrupt motoneuron pathfinding, ruling out an autocrine
mechanism. By contrast, expression of BaboI and PutI receptors
in motoneurons effectively phenocopies daw-
(Fig. 6,
Table 3), suggesting that axon
guidance defects could arise from the inability of motoneurons to respond to a
paracrine Daw signal. Interestingly, the retrograde Gbb/BMP signal transduced
by Wit/Tkv and Mad that regulates synapse morphology and function in larval
motoneurons (Aberle et al.,
2002; Marques et al.,
2002), shows minimal crosstalk despite acting in the same tissue.
Disruption of BMP signaling, by expression of TkvI in motoneurons
(Fig. 5C) or mutations in
wit (Marques et al.,
2002), does not affect axon guidance although it affects
neuromuscular junction (NMJ) function.
Daw functions as a permissive signal to modulate response to other guidance cues
An important question is whether Daw functions as an instructive cue that
provides directional information, or as a permissive factor that promotes axon
outgrowth. Several lines of evidence argue against an instructive role. First,
axon pathfinding does not require restricted expression of daw.
Guidance defects associated with daw mutants can be rescued by
daw expression in sites of endogenous transcription, and ectopically
in motoneurons (Table 4).
Second, in daw mutants, axons do not extend into inappropriate areas
or show ectopic branching, phenotypes typical of mutations in Sema-2a,
Netrin-A and Netrin-B that provide spatial guidance cues or
target recognition (Harris et al.,
1996; Mitchell et al.,
1996; Winberg et al.,
1998a). Finally, misexpression of Daw did not cause mistargeting
of axons, indicating no apparent spatial sensitivity to Daw. Our data are
therefore consistent with a permissive role in which Daw enables and/or
modulates the response of the growth cone to other restricted cues.
Potential targets of Daw signaling
Both daw and Smad2 mutants display similar errors in
pathfinding, suggesting that daw acts at the transcriptional level by
altering the expression of one or more molecules that regulate growth cone
response or motility. We found no evidence for cell fate changes in the
embryonic nervous system of daw mutants; and pathfinding defects can
be rescued using OK6-Gal4 that initiates expression in motoneurons at stage 15
(L.P. and K.A., unpublished), well after neuronal cell fates are specified
(Table 4). Furthermore, we find
no guidance errors in daw third-instar larvae. These results argue
that motoneurons are correctly specified and that the embryonic pathfinding
errors are compensated by larval stages.
Daw signaling could act on a wide range of transcriptional targets in the
neuron. Mutations in several genes that function as guidance cues, such as
plexin A, Sema-1a and side, show ISNb and SNa phenotypes
similar to daw mutants. Thus daw could alter the activity of
or the response to these guidance cues. Plexin A and Sema-1a are expressed in
neurons and mediate local repulsion
(Winberg et al., 1998b). Side,
a muscle-derived attractant is unlikely to be directly regulated
(de Jong et al., 2005;
Sink et al., 2001), however
components involved in the response to side could be downstream of
daw. Interestingly, overexpression of the IgCAM Fas2 that promotes
axon fasciculation also results in stalling defects reminiscent of the daw
phenotype (Lin and Goodman,
1994). Thus, Daw could act on Fas2 or Beat-Ia, which potentially
downregulates Fas2 in motoneurons
(Fambrough and Goodman, 1996),
to decrease adhesiveness at specific choice points in response to cues
directing defasciculation. Other possible targets are the RPTPs that affect
fasciculation and outgrowth. Whereas the Lar-null phenotype is
significantly stronger than that of daw, the combinatorial loss of
RPTP10D, RPTP69D and RPTP99A mimics the loss of Daw activity in ISNb and SNa
pathways (Desai et al., 1996;
Desai et al., 1997;
Krueger et al., 1996;
Sun et al., 2001). Finally,
daw mutants show phenotypic overlap with mutations in the
actin-microtubulecrosslinking proteins Pod1 and Shot, and in the actin-binding
protein Profilin, which are required for axon outgrowth
(Huber et al., 2003;
Lee et al., 2000;
Rothenberg et al., 2003;
Wills et al., 1999). This
raises the possibility that Daw signaling could control the expression or
activity of genes involved in regulating cytoskeletal dynamics. Future
epistasis studies will resolve whether Daw acts in conjunction with, or
parallel to, known pathways that regulate axon fasciculation and
extension.
The consequences of mutations in daw are often less severe and
limited to a subset of axon pathways affected by the genes discussed above,
suggesting that Daw could in part act redundantly with other proteins.
Alternatively, daw activity could be spatially restricted to select
axon pathways by localized expression of receptors, coreceptors or other
pathway components. For example, in the Drosophila CNS, only axons
expressing the Derailed receptor are sensitive to the Wnt5 repulsive cue, and
are hence directed away from the posterior into the anterior commissure
(Yoshikawa et al., 2003).
Alternatively, receptor expression could be dynamically modulated, as seen in
the downregulation of Robo in commissural neurons by transient expression of
Commissureless, which results in local insensitivity to Slit at the midline
(Keleman et al., 2002).
Although mRNA for the Daw-receptors Babo and Put can be detected throughout
the VNC (Fig. 5A,B)
(Childs et al., 1993), it
remains to be seen whether the proteins are enriched in a subset of growth
cones, restricting the response to Daw. A further possibility is that the
activity of the ligand itself could be spatially regulated. A recent study has
shown that mutations in tolloid-related (tlr;
tolkin - Flybase) that encodes a metalloprotease of the BMP1/Tolloid
family (Nguyen et al., 1994),
display persistent pathfinding defects
(Meyer and Aberle, 2006). In
an accompanying manuscript (Serpe and
O'Connor, 2006), tlr is shown to be required for
activation of a latent Daw complex. Intriguingly, we find that daw/+;
tlr/+ embryos show pathfinding defects consistent with a functional link
between the two genes (L.P. and K.A., unpublished). However, localized
activation of Daw by Tlr appears unlikely because Tlr is present in the
hemolymph and circulates throughout the embryo. Finally, Daw interaction with
the extracellular matrix or HSPGs could result in localized presentation of
the ligand to the extending growth cone. HSPGs are known to modulate BMP
activity by affecting ligand stability and receptor interaction
(Hacker et al., 2005). An
intriguing possibility is that in addition to functioning as ligands for Lar
(Fox and Zinn, 2005;
Johnson et al., 2006), HSPGs
could also function in axon guidance by enhancing Daw signaling.
TGF-ß signaling and axonal pathfinding
This study provides the first evidence that an activin pathway, acting
through its transcription factor Smad2, can direct axon pathfinding. The
mechanism by which Daw functions stands in contrast to previous studies
implicating BMP/TGF-ß ligands in direct regulation of growth cone
motility independent of a nuclear response. In vertebrates, BMP7/GDF7
heterodimers secreted by the spinal cord roof plate mediate repulsion of
commissural axons away from the dorsal midline
(Butler and Dodd, 2003).
Exposure to BMP7 for as little as 30 minutes resulted in growth cone collapse
in cultured neurons. BMP7 can also stimulate formation of dendritic arbors by
directly regulating the cytoskeleton. This Smad-independent effect requires
interaction of the BMP type-II receptor with LIMK1 that regulates the
actin-depolymerising factor cofilin
(Foletta et al., 2003;
Lee-Hoeflich et al., 2004),
and suggests a potential mechanism by which BMPs may influence growth cone
motility as well. In Drosophila, a BMP/Wit pathway acting through
LIMK1 promotes synapse stabilization at the NMJ although this is independent
of ADF/Cofilin, suggesting that at least part of this mechanism is conserved
(Eaton and Davis, 2005).
In C. elegans, the TGF-ß family member UNC-129 functions as a
target-derived chemoattractant for dorsally projecting motor axons
(Colavita et al., 1998).
UNC-129 is also likely to exploit an unconventional mechanism to direct
motoneuron guidance, as mutations in the single type-II receptor DAF-4 or the
Smad signaltransducer do not cause pathfinding defects. Thus TGF-ß family
members may utilize both canonical (as seen for Daw) and noncanonical
strategies to regulate neuronal guidance, depending on context. It remains to
be determined if an activin signaling pathway, comparable to Daw, plays a role
in vertebrate axon guidance. The finding that axons from ventrally-derived
retinal ganglion cells fail to enter the optic nerve head in
Bmpr1b-deficient mice implicates a conventional BMP
signal-transduction pathway in vertebrate growth cone guidance
(Liu et al., 2003).
Conversely, the recent finding that LIMK1 and cofilin have been implicated in
axon outgrowth in mushroom body neurons
(Ng and Luo, 2004), raises the
possibility that an activin/BMP ligand acting through Wit could initiate this
process in the larval brain.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Aberle, H., Haghighi, A. P., Fetter, R. D., McCabe, B. D., Magalhaes, T. R. and Goodman, C. S. (2002). wishful thinking encodes a BMP type II receptor that regulates synaptic growth in Drosophila. Neuron 33,545 -558.[CrossRef][Medline]
Altmann, C. R. and Brivanlou, A. H. (2001). Neural patterning in the vertebrate embryo. Int. Rev. Cytol. 203,447 -482.[Medline]
Bate, M. and Broadie, K. (1995). Wiring by fly: the neuromuscular system of the Drosophila embryo. Neuron 15,513 -525.[CrossRef][Medline]
Brummel, T., Abdollah, S., Haerry, T. E., Shimell, M. J.,
Merriam, J., Raftery, L., Wrana, J. L. and O'Connor, M. B.
(1999). The Drosophila activin receptor baboon signals through
dSmad2 and controls cell proliferation but not patterning during larval
development. Genes Dev.
13, 98-111.
Butler, S. J. and Dodd, J. (2003). A role for BMP heterodimers in roof platemediated repulsion of commissural axons. Neuron 38,389 -401.[CrossRef][Medline]
Childs, S. R., Wrana, J. L., Arora, K., Attisano, L., O'Connor,
M. B. and Massague, J. (1993). Identification of a
Drosophila activin receptor. Proc. Natl. Acad. Sci.
USA. 90,9475
-9479
Chisholm, A. and Tessier-Lavigne, M. (1999). Conservation and divergence of axon guidance mechanisms. Curr. Opin. Neurobiol. 9,603 -615.[CrossRef][Medline]
Colavita, A., Krishna, S., Zheng, H., Padgett, R. W. and
Culotti, J. G. (1998). Pioneer axon guidance by UNC-129, a C.
elegans TGF-beta. Science
281,706
-709.
Das, P., Inoue, H., Baker, J. C., Beppu, H., Kawabata, M., Harland, R. M., Miyazono, K. and Padgett, R. W. (1999). Drosophila dSmad2 and Atr-I transmit activin/TGFbeta signals. Genes Cells 4,123 -134.[Abstract]
de Jong, S., Cavallo, J. A., Rios, C. D., Dworak, H. A. and Sink, H. (2005). Target recognition and synaptogenesis by motor axons: responses to the sidestep protein. Int. J. Dev. Neurosci. 23,397 -410.[CrossRef][Medline]
Desai, C. J., Gindhart, J. G., Jr, Goldstein, L. S. and Zinn, K. (1996). Receptor tyrosine phosphatases are required for motor axon guidance in the Drosophila embryo. Cell 84,599 -609.[CrossRef][Medline]
Desai, C. J., Krueger, N. X., Saito, H. and Zinn, K. (1997). Competition and cooperation among receptor tyrosine phosphatases control motoneuron growth cone guidance in Drosophila. Development 124,1941 -1952.[Abstract]
Dickson, B. J. (2002). Molecular mechanisms of
axon guidance. Science
298,1959
-1964.
Eaton, B. A. and Davis, G. W. (2005). LIM Kinase1 controls synaptic stability downstream of the type II BMP receptor. Neuron 47,695 -708.[CrossRef][Medline]
Fambrough, D. and Goodman, C. S. (1996). The Drosophila beaten path gene encodes a novel secreted protein that regulates defasciculation at motor axon choice points. Cell 87,1049 -1058.[CrossRef][Medline]
Faure, S., Lee, M. A., Keller, T., ten Dijke, P. and Whitman, M. (2000). Endogenous patterns of TGFbeta superfamily signaling during early Xenopus development. Development 127,2917 -2931.[Abstract]
Foletta, V. C., Lim, M. A., Soosairajah, J., Kelly, A. P.,
Stanley, E. G., Shannon, M., He, W., Das, S., Massague, J. and Bernard, O.
(2003). Direct signaling by the BMP type II receptor via the
cytoskeletal regulator LIMK1. J. Cell Biol.
162,1089
-1098.
Fox, A. N. and Zinn, K. (2005). The heparan sulfate proteoglycan syndecan is an in vivo ligand for the Drosophila LAR receptor tyrosine phosphatase. Curr. Biol. 15,1701 -1711.[CrossRef][Medline]
Hacker, U., Nybakken, K. and Perrimon, N. (2005). Heparan sulphate proteoglycans: the sweet side of development. Nat. Rev. Mol. Cell Biol. 6, 530-541.[CrossRef][Medline]
Haerry, T. E., Khalsa, O., O'Connor, M. B. and Wharton, K. A. (1998). Synergistic signaling by two BMP ligands through the Sax and Tkv receptors controls wing growth and patterning in Drosophila. Development 125,3977 -3987.[Abstract]
Harris, R., Sabatelli, L. M. and Seeger, M. A. (1996). Guidance cues at the Drosophila CNS midline: identification and characterization of two Drosophila Netrin/UNC-6 homologs. Neuron 17,217 -228.[CrossRef][Medline]
Huber, A. B., Kolodkin, A. L., Ginty, D. D. and Cloutier, J. F. (2003). Signaling at the growth cone: ligand-receptor complexes and the control of axon growth and guidance. Annu. Rev. Neurosci. 26,509 -563.[CrossRef][Medline]
Johnson, K. G., Tenney, A. P., Ghose, A., Duckworth, A. M., Higashi, M. E., Parfitt, K., Marcu, O., Heslip, T. R., Marsh, J. L., Schwarz, T. L. et al. (2006). The HSPGs Syndecan and Dallylike bind the receptor phosphatase LAR and exert distinct effects on synaptic development. Neuron 49,517 -531.[CrossRef][Medline]
Jones, B. W., Fetter, R. D., Tear, G. and Goodman, C. S. (1995). glial cells missing: a genetic switch that controls glial versus neuronal fate. Cell 82,1013 -1023.[CrossRef][Medline]
Keleman, K., Rajagopalan, S., Cleppien, D., Teis, D., Paiha, K., Huber, L. A., Technau, G. M. and Dickson, B. J. (2002). Comm sorts robo to control axon guidance at the Drosophila midline. Cell 110,415 -427.[CrossRef][Medline]
Keshishian, H., Broadie, K., Chiba, A. and Bate, M. (1996). The drosophila neuromuscular junction: a model system for studying synaptic development and function. Annu. Rev. Neurosci. 19,545 -575.[CrossRef][Medline]
Krueger, N. X., Van Vactor, D., Wan, H. I., Gelbart, W. M., Goodman, C. S. and Saito, H. (1996). The transmembrane tyrosine phosphatase DLAR controls motor axon guidance in Drosophila. Cell 84,611 -622.[CrossRef][Medline]
Landgraf, M., Bossing, T., Technau, G. M. and Bate, M.
(1997). The origin, location, and projections of the embryonic
abdominal motorneurons of Drosophila. J. Neurosci.
17,9642
-9655.
Lee, S., Harris, K. L., Whitington, P. M. and Kolodziej, P.
A. (2000). short stop is allelic to kakapo, and encodes
rod-like cytoskeletal-associated proteins required for axon extension.
J. Neurosci. 20,1096
-1108.
Lee-Hoeflich, S. T., Causing, C. G., Podkowa, M., Zhao, X., Wrana, J. L. and Attisano, L. (2004). Activation of LIMK1 by binding to the BMP receptor, BMPRII, regulates BMP-dependent dendritogenesis. EMBO J. 23,4792 -4801.[CrossRef][Medline]
Lee-Hoeflich, S. T., Zhao, X., Mehra, A. and Attisano, L. (2005). The Drosophila type II receptor, Wishful thinking, binds BMP and myoglianin to activate multiple TGFbeta family signaling pathways. FEBS Lett. 579,4615 -4621.[CrossRef][Medline]
Letsou, A., Arora, K., Wrana, J. L., Simin, K., Twombly, V., Jamal, J., Staehling-Hampton, K., Hoffmann, F. M., Gelbart, W. M., Massague, J. et al. (1995). Drosophila Dpp signaling is mediated by the punt gene product: a dual ligand-binding type II receptor of the TGF beta receptor family. Cell 80,899 -908.[CrossRef][Medline]
Lin, D. M. and Goodman, C. S. (1994). Ectopic and increased expression of Fasciclin II alters motoneuron growth cone guidance. Neuron 13,507 -523.[CrossRef][Medline]
Littleton, J. T. and Bellen, H. J. (1994). Genetic and phenotypic analysis of thirteen essential genes in cytological interval 22F1-2; 23B1-2 reveals novel genes required for neural development in Drosophila. Genetics 138,111 -123.[Abstract]
Liu, J., Wilson, S. and Reh, T. (2003). BMP receptor 1b is required for axon guidance and cell survival in the developing retina. Dev. Biol. 256,34 -48.[CrossRef][Medline]
Lo, P. C. and Frasch, M. (1999). Sequence and expression of myoglianin, a novel Drosophila gene of the TGF-beta superfamily. Mech. Dev. 86,171 -175.[CrossRef][Medline]
Luo, L. (2002). Actin cytoskeleton regulation in neuronal morphogenesis and structural plasticity. Annu. Rev. Cell Dev. Biol. 18,601 -635.[CrossRef][Medline]
Marques, G., Bao, H., Haerry, T. E., Shimell, M. J., Duchek, P., Zhang, B. and O'Connor, M. B. (2002). The Drosophila BMP type II receptor Wishful Thinking regulates neuromuscular synapse morphology and function. Neuron 33,529 -543.[CrossRef][Medline]
Massague, J. (1998). TGF-beta signal transduction. Annu. Rev. Biochem. 67,753 -791.[CrossRef][Medline]
Mehler, M. F., Mabie, P. C., Zhang, D. and Kessler, J. A. (1997). Bone morphogenetic proteins in the nervous system. Trends Neurosci. 20,309 -317.[Medline]
Meyer, F. and Aberle, H. (2006). At the next stop sign turn right: the metalloprotease Tolloid-related 1 controls defasciculation of motor axons in Drosophila. Development 133,4035 -4044.
