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First published online 13 February 2008
doi: 10.1242/dev.012989
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Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA.
* Author for correspondence (e-mail: butlersj{at}usc.edu)
Accepted 9 January 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Axon guidance, Bone morphogenetic proteins, Commissural neurons, Morphogen, Spinal cord
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Salie
et al., 2005). The ability of morphogens to act as axon guidance
cues was first shown for the commissural neurons
(Augsburger et al., 1999), a
class of dorsal sensory interneurons in the developing spinal cord
(Holley, 1982;
Dodd et al., 1988).
Commissural neurons differentiate adjacent to the dorsal midline in response
to inductive signals from Bone Morphogenetic Proteins (BMPs) present in the
roof plate (RP) (Liem et al.,
1997; Lee et al.,
1998). They then extend axons away from the RP, in a ventral and
circumferential route through the dorsal spinal cord
(Holley, 1982;
Oppenheim et al., 1988). Our
previous studies have shown that this initial trajectory of commissural axons
is also directed by the activity of BMPs, exerting their function as a
heterodimer of BMP7 and Growth/Differentiation Factor 7 (GDF7)
(Augsburger et al., 1999;
Butler and Dodd, 2003). Thus,
BMPs act as a classic feed-forward signal, directing different outcomes at
different stages of commissural neuronal development: BMPs first act as
morphogens to specify commissural cell fate and then as axon guidance cues to
direct commissural axons away from the RP.
The ability of the BMPs to act as axon guidance molecules appears to be an
activity common to other morphogens. Sonic Hedgehog (Shh) and members of the
Wnt and Fibroblast Growth Factor (FGF) families also act as axon guidance
molecules (Irving et al.,
2002; Charron et al.,
2003; Lyuksyutova et al.,
2003), suggesting a model in which the same factors pattern the
diversity of both cell fate and axonal connectivity within the nervous system.
These studies suggest that a single factor can specify unexpectedly diverse
activities for developing neurons, but they do not resolve how this process is
achieved mechanistically. Morphogens specify cell fate over many hours by
initiating global changes in the transcriptional status of the cell
(Tabata and Takei, 2004). By
contrast, axon guidance cues locally activate signal transduction pathways in
the axonal growth cone (Dickson,
2002), resulting in the rapid reorganization of the cytoskeleton
(Dodd and Jessell, 1988;
Tessier-Lavigne and Goodman,
1996) in a process that is independent of the nucleus
(Campbell and Holt, 2001). It
remains unclear how cells distinguish between the cell fate specification and
axon guidance activities of morphogens. One possibility is that the diverse
activities of morphogens are transduced by signalling through distinct signal
transduction pathways. Alternatively, morphogens could signal through the same
receptor and signalling components, with the outcome being determined by the
context in which the signal is perceived. Studies addressing this question
have suggested that morphogens employ both of these strategies to mediate
their diverse activities. For example, distinct receptor complexes appear to
translate the guidance and inductive activities of Shh. Shh has recently been
shown to direct commissural axon guidance decisions by activating either the
non-canonical Boc (Okada et al.,
2006) or Hip (Bourikas et al.,
2005) receptors, whereas Patched and Smoothened (Smo) mediate the
inductive activities of Shh (Nybakken and
Perrimon, 2002). By contrast, the canonical FGF receptors appear
to be important in retinal axon guidance
(Brittis et al., 1996;
McFarlane et al., 1996). For
the Wnts, a combinatorial model is emerging in which the canonical Wnt
receptor Frizzled interprets the attractive axonal responses to Wnts, whereas
repulsive Wnt guidance cues are transduced by the atypical receptor Ryk
(reviewed by Bovolenta et al.,
2006).
Here, we assess the mechanism by which the activity of the BMP-mediated RP
chemorepellent is transduced. The canonical BMP receptor (BMPR) complex
consists of type I and II serine/threonine kinases
(Ebendal et al., 1998) that,
upon ligand binding, phosphorylate the receptor-regulated Smads (R-Smads), a
class of intracellular signalling effectors
(Heldin et al., 1997). The
specification of cell fate by the BMPs is thought to be mediated by the
ability of the Smad complex to regulate the transcription of target genes,
following its translocation to the nucleus
(Kretzschmar and Massague,
1998). However, it is unlikely that BMPs guide axons by altering
the transcriptional status of the cell; the direct application of BMPs to
commissural growth cones rapidly causes their collapse, suggesting that the
BMP guidance signal is transduced locally in the growth cone by a
non-transcriptionally based mechanism
(Augsburger et al., 1999).
Additionally, BMP ligands may have differential activities. BMP homodimers
important for inductive activity are not the principal mediators of guidance
activity; rather, this function is carried out by a BMP7:GDF7 heterodimer
(Butler and Dodd, 2003).
|
);
however, the type I BMPRs have been largely shown to function redundantly in
the specification of cell fate (Murali et
al., 2005; Yoon et al.,
2005). In particular, the dorsal-most neurons in the mouse spinal
cord are only lost in BmprIa-/-; BmprIb-/-
double mutants (Wine-Lee et al.,
2004) and the constitutive activation of either BMPRIA or BMPRIB
in the chick spinal cord leads to the increased production of dorsal neurons
(Timmer et al., 2002). Here,
we use both gain- and loss-of-function approaches to show that the type I
BMPRs also mediate commissural axon outgrowth and guidance. However, whereas
both type I BMPRs contribute to the assignment of dorsal cell fates in the
spinal cord, axon guidance activity is principally mediated by BMPRIB, as only
BMPRIB is both necessary and sufficient to mediate the known activities of the
BMP component of the RP chemorepellent. These results suggest that the
differential activation of particular BMPR complexes distinguishes between the
inductive and guidance activities of the BMP morphogen. |
MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and Akiyama et al.
(Akiyama et al., 1997).
Expression constructs containing either the caBMPRs or farnesylated EGFP
(fGFP, Invitrogen) fused to the Math1 enhancer were generated by
inserting a 1.7 kb Math1 enhancer fragment (Tg9) into the BGZA vector
(Helms et al., 2000) and
replacing the lacZ reporter gene with either fGFP or a cassette of
caBMPR-IRES-fGFP.
Hamilton Hamburger (HH) stage 11 to 15
(Hamburger and Hamilton, 1992)
White Leghorn chick embryos (AA Laboratory Eggs) were injected with 0.2-2.0
µg/µl plasmid DNA solutions, and electroporated and processed as
previously described (Briscoe et al.,
2000). For the Math1 expression constructs, low concentrations
(0.2 µg/µl) of the plasmid were used to ensure that ectopic gene
expression was spatially restricted to the dorsal spinal cord. All statistical
analyses were performed using a one-tailed Student's t-test.
Generation and analysis of mutant mice
All mice were of the same inbred genetic background (129/Sv) and the
embryos were genotyped by PCR (Yi et al.,
2000). Whole-mount fillet preparations and transverse sections of
the spinal cord from wild-type or mutant E11.5 embryos were prepared and
quantified as described previously (Butler
and Dodd, 2003).
Explant cultures
Explants of E11 rat roof plate and E10.5 mouse dorsal spinal cord were
dissected, cultured and immunostained as previously described
(Augsburger et al., 1999).
Immunohistochemistry
The following antibodies were used: 
Tag1
(Dodd et al., 1988) (mAb 4D7)
at 1:6; Lh2a/Lh2b (Liem et al.,
1997) (L1, rabbit pan (p) Lh2) at 1:1000; Isl1/Isl2
(Tsuchida et al., 1994) (K5,
rabbit pIsl) at 1:1000; rabbit Math1
(Helms and Johnson, 1998) at
1:500; rabbit Axonin1 (Ruegg et
al., 1989) at 1:2000; HA (mAb) at 1:2000 (Covance);
phosphorylated (phos) Smad1/5/8 (Cell Signaling Technology) at 1:1000;
Cre mAb at 1:2000 (Covance); and sheep GFP at 1:2000
(Biogenesis). Cy3-, Cy5- or FITC-coupled secondary antibodies were used
(Jackson Laboratories). Images were collected on Zeiss LSM510 confocal and
Axiovert 200M microscopes.
In situ hybridization
BmprIa and BmprIb DIG-labeled riboprobes were hybridized
to 20-µm thick cryosections of E11.5 fresh-frozen mouse tissue as
previously described (Schaeren-Wiemers and
Gerfin-Moser, 1993).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Roelen et al.,
1997). To determine whether the type I BMPRs show regionally
specific expression in the spinal cord, the expression patterns of both BMPRIA
and BMPRIB were analyzed at embryonic mouse stage (E) 11.5 when commissural
axiogenesis is ongoing. BMPRIA is expressed throughout the ventricular zone
(VZ) in the spinal cord, consistent with its role directing cell fate in
neuronal progenitors (Fig. 1A),
and is absent from the mantle layer (dashed lines,
Fig. 1A). By contrast, BMPRIB
has a more spatially restricted distribution: it is expressed in the dorsal
and intermediate VZ (Fig. 1B),
as well as in the dorsal-most population of postmitotic neurons in the mantle
layer (dashed lines, Fig. 1B).
These neurons overlap with those labeled by antibodies against Math1 (Atoh1 -
Mouse Genome Informatics), a marker of early-born commissural neurons
(arrowheads, Fig. 1C,D)
(Helms and Johnson, 1998).
This distribution pattern indicates that, of the type I BMPRs, only BMPRIB is
present in commissural neurons during axiogenesis.
Constitutively active type I BMPRs affect dorsal cell fate only in early stages of chick development
To assess the role of the type I BMPRs in commissural axon guidance, both
gain-of-function and loss-of-function approaches were taken. For the
gain-of-function studies, we examined the consequence of constitutively
activating the type I BMPRs in commissural neurons in chick embryos. Previous
studies have shown that ectopically activating either of the type I BMPRs in
the chick spinal cord resulted in increased numbers of dorsal neurons
(Timmer et al., 2002),
suggesting that the type I BMPRs transduce the RP-derived BMP morphogen
signal. Thus, it was crucial in our studies to determine whether any effect of
modulating BMPR activity on the trajectory of commissural axons was due to a
primary defect in axon guidance or was a secondary consequence of altered
inductive signalling. To address this question, we determined whether the
effect of constitutively active (ca) type I BMPRs on dorsal neural identity is
temporally restricted. The CMV enhancer was used to direct the expression of
EGFP (CMV::GFP) in combination with either haemagglutinin (HA)-tagged caBMPRIA
or caBMPRIB (CMV::caBMPRIA, CMV::caBMPRIB). These constructs were introduced
into the chick spinal cord at different developmental stages by in ovo
electroporation (Swartz et al.,
2001). The status of cellular identity in the chick spinal cord
following electroporation was determined in Hamilton Hamburger (HH) stage
22/23 embryos using a panel of markers for spinal neuronal progenitors and
early differentiated neurons (Fig.
2M). These markers included antibodies against pLh2, which labels
postmitotic commissural (dI1) neurons
(Liem et al., 1997), and pIsl,
which labels association (dI3) neurons and motoneurons (MNs)
(Tsuchida et al., 1994).
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), the
misexpression of either caBMPR in HH stage 11/12 chick embryos resulted in an
alteration in the cellular identity of the spinal cord (see Fig. S1 in the
supplementary material), presumably because the constitutive activation of
either type I BMPR leads to both the induction of cells with dorsal fates and
the suppression of the ventral cell fates. By contrast, when the expression
constructs were electroporated into HH stage 14/15 chick embryos, the fate of
the dorsal spinal cord was unaffected (Fig.
2A-H). Ventral cellular identity was, nevertheless, partially
affected; the number of pIsl+ MNs appeared to be slightly decreased
on the electroporated side (Fig.
2E-H), although no loss of Nkx2.2+ V3 interneurons was
observed (data not shown). Both type I caBMPR constructs were functional: at
all stages tested, the BMP-specific R-Smads, Smad1/Smad5/Smad8, were activated
to the same extent after the electroporation of either caBMPR
(Fig. 2I-M; see also Fig. S1I-L
in the supplementary material). To quantify these results further, we compared the number of dI1 and dI3 interneurons on the electroporated and non-electroporated sides of the spinal cord (Fig. 2N,O). The electroporation of either CMV::caBMPRIA or CMV::caBMPRIB into stage 11/12 embryos resulted in a significant increase in the number of either dI1 or dI3 neurons (see Fig. S1 in the supplementary material; Fig. 2O). By contrast, when these constructs were electroporated into stage 14/15 embryos, similar numbers of dI1 and dI3 neurons were seen on both sides of the spinal cord. These results suggest that only spinal tissue at the earliest stages of chick neural tube development is competent to respond to the type I caBMPRs and to adopt a dorsal cellular fate. This competence is lost by stage 14, when commissural axiogenesis is beginning. Taken together, these results suggest that the type I BMPRs no longer mediate the inductive activities of the BMPs by this point in development.
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) on the
trajectory of chick commissural axons. Chick commissural axons have an
indistinguishable trajectory from rodent commissural axons in the transverse
plane of the spinal cord and are responsive to the same axon guidance cues
(Holley, 1982;
Kennedy et al., 1994;
Serafini et al., 1996). If
local activation of the type I BMPRs in commissural neurons translates the BMP
gradient from the RP into directed axonal growth away from the dorsal midline,
then a neuron expressing a caBMPR should perceive an altered gradient of BMPs
and will extend an axon along an aberrant trajectory.
Constructs containing HA-tagged BMPRIA or BMPRIB under the control of the
CMV enhancer were electroporated along with a CMV::GFP construct into the
spinal cords of stage 14/15 chick embryos, which were then analyzed at stage
22-25 when many commissural axons have reached the floor plate (FP).
Commissural axons were further visualized using antibodies against Axonin1,
the chick homolog of Tag1, which labels both commissural axons and early-born
MNs (Ruegg et al., 1989).
Electroporation of chick embryos with the CMV::caBMPRIA construct had no
effect on either GFP+ or Axonin1+ axon trajectories
(Fig. 3A-D). GFP+
axons projected normally, either exiting from the spinal cord or crossing the
FP (arrowhead, Fig. 3B).
Similarly, Axonin1+ axons on the electroporated side of the spinal
cord behaved identically to those on the non-electroporated side, projecting
ventrally, away from the RP (Fig.
3C,D). By contrast, the trajectories of both GFP+ and
Axonin1+ axons were severely compromised after misexpression of
caBMPRIB (Fig. 3E-H). The
electroporated axons exhibited two behaviours: some Axonin1+ axons
were mispolarized, extending medially into the VZ (arrows,
Fig. 3G,H); others appeared to
stall as they approached the ventral midline (open arrowhead,
Fig. 3G). Supporting this
latter observation, no dorsally derived GFP+ axons made the ventral
contralateral projection across the FP (arrowhead,
Fig. 3F), although the
ventrally derived GFP+ motor axons exited the spinal cord
normally.
These results suggest that the type I BMPRs differ in their abilities to
mediate commissural axon guidance. This divergence in function was unexpected,
given that BMPRIA and BMPRIB have significantly overlapping functions in other
systems, in particular, the specification of dorsal cell fate in the
developing spinal cord (Wine-Lee et al.,
2004). However, a caveat in these experiments is that caBMPRIB was
misexpressed throughout the spinal cord, making it possible that the axon
guidance defects seen resulted from non-autonomous alterations in the
properties of the tissue surrounding the commissural neurons. Thus, it was
crucial to examine the effect of expressing the type I caBMPRs solely in
postmitotic commissural neurons. Towards this end, we generated constructs in
which either farnesylated (f) GFP, caBMPRIA or caBMPRIB were expressed under
the control of the Math1 enhancer, which drives gene expression specifically
in early-born commissural neurons in the spinal cord
(Helms et al., 2000;
Lumpkin et al., 2003). Both
the Math1::caBMPRIA and Math1::caBMPRIB constructs contained an IRES-fGFP
reporter to visualise the trajectories of the electroporated commissural
axons.
The electroporation of either the control Math1::fGFP vector or the Math1::caBMPRIA-IRES-fGFP construct had no effect on the trajectory of either Axonin1+ or GFP+ axons; the GFP+ axons projected normally to the FP (arrowheads, Fig. 4B,D,G). By contrast, electroporated Axonin1+ GFP+ axons in the Math1::caBMPRIB-IRES-fGFP embryos projected in a similar manner to the axons electroporated with CMV::caBMPRIB. GFP+ axons either misprojected medially into the VZ (arrow, Fig. 4F) or they stalled above the developing MN column (arrowheads, Fig. 4F,H), phenotypes consistent with BMPRIB transducing both directional and outgrowth information for commissural axons. The extent of the axon outgrowth defect was quantified by determining the percentage of electroporated commissural neurons that had extended axons to the midpoint of the dorsal (MD), intermediate (INT) or ventral (MV) regions of the spinal cord or to the FP (Fig. 4J). In Math1::fGFP embryos, 62.2%±1.5 of GFP+ neurons had extended axons to the MD line by stage 22/23 (Fig. 4I). Commissural axiogenesis is still ongoing at this stage, thus axon outgrowth evenly decreased with distance from the RP, with the majority (55%±1.4) of GFP+ axons having reached the MV region. In embryos electroporated with Math1::caBMPRIB-IRES-fGFP at the same stage, 48.7%±1.7 of commissural neurons had extended axons (Fig. 4I). However, in contrast to the control, there was a precipitous drop in the number of commissural axons extending past the INT line, with only 22.6%±1.0 of GFP+ axons now reaching the MV region. Taken together, these results suggest that constitutively activating BMPRIB, but not BMPRIA, in commissural neurons profoundly affects their ability to interpret the environment, resulting in both guidance and outgrowth defects.
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). Rather, these neurons were lost only in BmprIa;
BmprIb double mutant embryos
(Wine-Lee et al., 2004),
suggesting that any defects in axon guidance observed in the absence of either
BMPRIA or BMPRIB do not result from a failure of dorsal neural
differentiation. Mice mutant for BmprIb are viable
(Yi et al., 2000;
Yi et al., 2001); however, the
BmprIa mutation is lethal
(Mishina et al., 1995),
necessitating the use of a conditional allele of BmprIa
(BmprIaflox) (Mishina
et al., 2002). Tissue-specific recombination of BmprIa
was achieved by mating the BmprIaflox line to transgenic
mice expressing cre recombinase under the control of the Math1
enhancer (Matei et al., 2005).
The Math1 enhancer drives the expression of cre in postmitotic
commissural neurons (Fig.
5E,F), resulting in Cre-mediated recombination by stage E10
(Matei et al., 2005), the
stage after cell fate specification, but before the onset of commissural
axiogenesis. Thus, it was possible to determine the requirement for each of
the type I BMPRs in commissural axon guidance without the complicating effects
from disruptions in cell fate.
The trajectory of commissural axons was first assessed in transverse
sections of wild-type (Fig.
5A), Math1:cre;BmprIaflox/flox
(Fig. 5B) and
BmprIb-/- (Fig.
5C) E11.5 embryos. In all three cases, commissural axons extended
in a highly polarized manner away from the RP. However, in
BmprIb-/- embryos, a small population of commissural axons
was mispolarized medially towards the lumen (arrowhead,
Fig. 5C). To quantify this
phenotype, the number of pLh2+ postmitotic commissural neurons that
extend a Tag1+ axon medially was determined in sections of
wild-type and BmprIb-/- spinal cords taken from the same
axial levels. In wild-type embryos, 0.1%±0.1 of commissural axons
extended aberrantly. By contrast, 1.7%±0.4 of commissural axons were
mispolarized in BmprIb-/- embryos, a figure comparable to
the number of mispolarized commissural axons in Bmp7 mutant embryos
(Butler and Dodd, 2003). This
result suggests that the loss of BMPRIB, but not BMPRIA, results in a
perturbation of the commissural axon trajectory in vivo.
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|
) and
Math1:cre;BmprIaflox/flox
(Fig. 6B) commissural axons
very rarely project into the RP and never cross the RP. However, the polarity
of the commissural axon trajectory was perturbed in
BmprIb-/- fillet preparations
(Fig. 6C,D). A significantly
increased number of commissural axons extended into the spinal cord (open
arrowheads, Fig. 6C) and were
occasionally seen to cross the RP (arrowhead,
Fig. 6D'). These
mispolarization phenotypes were similar to those seen in fillet preparations
from BMP mutant embryos (Butler and Dodd,
2003); however, they occurred at a lower frequency. To assess
whether BMPRIA has an activity that can compensate for the loss of BMPRIB, we
examined fillet preparations taken from E11.5
Math1:cre;BmprIaflox/flox; BmprIb-/-
mutant embryos (Fig. 6E,F). In
these fillets, the extent of commissural axon mispolarization was now found to
be comparable to that seen in either Bmp7 or Gdf7 mutants
(see Fig. S2 in the supplementary material). Taken together, these results
suggest the type I BMPRs are required to transduce the BMP component of the RP
chemorepellent in vivo. BMPRIB appears to be the principal type I receptor
that mediates the axon guidance activity of the BMPs, with BMPRIA necessary
for commissural axon orientation only in the absence of BMPRIB.
|
;
Butler and Dodd, 2003) that can
be used to measure the extent to which commissural axons respond to the RP
chemorepellent. Explants of the dorsal spinal cord were dissected from E10.5
wild-type, Math1:cre;BmprIaflox/flox and
BmprIb-/- mouse embryos. The commissural axon trajectory
was then challenged by placing a RP explant, taken from E11 rat embryos, in
contact with one of the lateral edges of the dorsal spinal explant
(Fig. 7D). Commissural growth
cones extending adjacent to the appended RP grow under both its influence and
that of the endogenous RP, and the extent to which they are reoriented under
these circumstances can be quantified. Consistent with previous observations
(Butler and Dodd, 2003), E10.5
wild-type mouse commissural axons were reoriented by a rat RP explant
(Fig. 7A,A'), with an
average reorientation angle of 21.8°±2.0
(Fig. 7E).
Math1:cre;BmprIaflox/flox commissural axons were
deflected to a similar extent (Fig.
7B,B'), with an average reorientation angle of
20.4°±2.0 (Fig. 7E).
By contrast, BmprIb-/- commissural axons were severely
compromised in their ability to reorient away from the RP explant
(Fig. 7C,C'). The average
angle by which BmbrIb-/- commissural axons are reoriented
is reduced to 9.55°±1.8 (Fig.
7E), which is statistically identical to the reorientation angles
seen when RP explants taken from Bmp7-/-
(9.26°±1.9) or Gdf7-/- (8.23°±1.4)
mutant mice were used to challenge wild-type rat commissural axons (see Fig.
S3 in the supplementary material) (Butler
and Dodd, 2003). Thus, removing BMPRIB, but not BMPRIA, from
commissural neurons has the same biological consequence as removing the BMPs
from the RP, an observation that strongly suggests that BMPRIB is the sole
type I receptor that mediates the ability of the BMP ligand to deflect
commissural axons in the reorientation assay. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Only BMPRIB is sufficient to disrupt commissural axon guidance
Our gain-of-function studies have demonstrated that the activities of the
type I BMPRs can be temporally separated. Thus, neural progenitors appear to
have a limited period during early spinal development in which they are
competent to distinguish the BMPs as morphogens. It remains unclear how dorsal
neural progenitors modulate their ability to respond to the BMP signal. The
downregulation of BmprIa in postmitotic commissural neurons
(Fig. 1) suggests that the
competence to respond to the BMPs as morphogens could depend on the presence
of BMPRIA. However, this model cannot be the case, because misexpressing
BmprIa later in spinal development has no effect on dorsal cell
induction (Fig. 2). The
presence of BmprIa in the ventral spinal cord is intriguing, given
that BMP signalling has been shown to antagonize Shh signalling in the
specification of ventral cell fates (Liem
et al., 2000). However, it remains unclear whether BMPRIA can
modulate ventral cell identity.
|
). Thus,
the electroporated commissural growth cones presumably perceive a
foreshortened gradient of BMPs, rather than the uniform distribution of
BMPs.
We also observed an unexpected defect in axon outgrowth following in ovo
electroporation with caBMPRIB: commissural axons stalled upon reaching the
ventral spinal cord. This defect does not appear to be a general delay in axon
outgrowth because commissural axons did not grow uniformly more slowly as they
projected ventrally around the spinal cord. Rather, there was a sharp decline
in the number of caBMPRIB+ axons projecting beyond the dorsal
spinal cord, suggesting that these axons stalled upon reaching the ventral
spinal cord. The basis for stalled axon outgrowth remains unclear. The
RP-derived BMPs may signal outgrowth information to commissural neurons.
Alternatively, caBMPRIB+ axons may be compromised in their ability
to respond to Shh and/or other attractive signals emanating from the FP. The
elevated levels of BMP signalling achieved in caBMPRIB+ axons may
antagonize Shh signalling, as has been shown for cell fate decisions
(Liem et al., 2000), thus
affecting the ability of commissural axons to respond to signals from the
FP.
Differential requirements for the type I BMPRs in commissural axon guidance
The results from the gain-of-function studies suggest that commissural
axons are guided away from the dorsal midline by the asymmetric activation of
BMPRIB within the commissural growth cone. This model predicts that
commissural growth cones will be similarly misguided by the uniform presence
of BMPs, i.e. after either the constitutive activation of BMPRIB, or the loss
of graded BMP signaling in the absence of either the ligand or relevant
receptor. Supporting this prediction, commissural axons in BmprIb
single mutants and BmprIa; BmprIb double mutants showed
mispolarization defects similar to those observed in the gain-of-function
studies.
A further prediction of the loss-of-function studies is that the loss of
the receptor that mediates the BMP component of the RP repellent will result
in comparable phenotypes to those seen in Bmp7 and Gdf7
mutants. In our previous work, we showed that the BMPs are required for the
ability of the RP to reorient commissural axons in vitro, and to establish the
polarized growth of commissural axons away from the RP in vivo
(Augsburger et al., 1999;
Butler and Dodd, 2003). In this
study, the absence of the type I BMPRs from commissural neurons phenocopies
the loss of either BMP gene from the RP. The in vitro reorienting activity of
the RP is transduced solely by BMPRIB, and BMPRIB appears to be the principal
receptor that mediates the BMP component of the RP in vivo, with BMPRIA
supplying a compensatory activity in the absence of BMPRIB. Only small effects
on commissural axon guidance were seen in our analysis of loss-of-function
mutations in either the BMP genes (Butler
and Dodd, 2003) or the type I BMP receptor genes (this study).
However, it is not unusual that the loss of key axon guidance signals in vivo
results in guidance defects that are either weak or transient, presumably
because of the presence of other redundant signals. Thus, the activities
revealed in in vitro assays, where such compensatory signals are not present,
may be a more accurate indication of the role of an axon guidance cue or
receptor than is revealed by loss-of-function genetic studies. Taking the in
vivo and in vitro studies together, these data strongly suggest that BMPRIB is
the crucial guidance receptor that translates BMP chemorepellent signals into
the directed movement of commissural axons away from the dorsal midline
(Fig. 8B).
The nature of the compensatory activity from BMPRIA remains unclear. BMPRIA alone is neither necessary nor sufficient as a guidance receptor for commissural axons. Thus, BMPRIA has either a very weak activity as a guidance receptor, or the compensatory activity of BMPRIA is a secondary effect of the role of dorsal cell fate specification in the assignment of neuronal polarity. Supporting this latter idea, BmprIa is not expressed in postmitotic commissural neurons and BMPRIA is required only in the absence of BMPRIB, consistent with the specification of cell fate being a redundant shared activity of BMPRIA and BMPRIB. Preliminary analysis has suggested that the distribution of BmprIa is not altered in BmprIb mutants (K.Y. and S.J.B., unpublished). Thus, the phenotypes seen in either the BmprIa; BmprIb double mutants or the BMP single mutants may be the result of defects both in neuronal polarity and axon guidance.
Differential roles of BMPRIA and BMPRIB in cell fate specification and axon guidance
In summary, our studies have suggested that the known activities of the BMP
guidance cue in the RP can be accounted for by signalling through the
canonical BMP signal transduction pathway. However, the type I BMPRs diverge
functionally in their ability to translate the inductive and guidance
activities of the BMPs. The specification of cell fate by the BMPs is a shared
activity of both type I BMPRs, whereas commissural axon guidance is
predominantly mediated by only one of the type I BMPRs, BMPRIB
(Fig. 8). The extent to which
BMPRIB mediates guidance decisions elsewhere in the developing nervous system
remains to be determined. However, studies showing that BMPRIB is required for
axon targeting in the developing retina
(Liu et al., 2003) suggests
BMPRIB may have a widespread role transducing BMP guidance signals.
How does BMP signalling result in two such different outcomes during
development? One possibility is the type I BMPRs are differentially activated
by particular BMP ligands. Thus, BMP homodimers direct cell fate decisions by
activating the shared property of the type I BMPRs, whereas the BMP7:GDF7
heterodimer reorients commissural axons by signalling through a unique
property of BMPRIB (Fig. 8).
Such differential signalling is then translated into a particular outcome by
the activation of the relevant second messenger intermediate. The morphogenic
activity of the BMPs is thought to be transduced by the Smad complex acting as
transcriptional regulators (Massague et
al., 2005). Additionally, BMP signaling has been shown to control
the activation status of Lim kinase1 (Limk1), a direct regulator of cofilin
(Foletta et al., 2003;
Lee-Hoeflich et al., 2004).
Recent studies in vitro have shown that a gradient of BMP7 can regulate actin
dynamics in Xenopus laevis growth cones by controlling the activity
of cofilin (Wen et al., 2007).
However, it remains to be determined which second messenger is relevant for
commissural axon guidance in vivo. BMPRIB could activate a different second
messenger to locally reorganize the cytoskeleton, such as Limk1, or the Smad
complex could have a novel role outside of the nucleus. The Smad complex has
not been previously shown to be active in the cytoplasm, although it is
intriguing that a neomorphic mutation in Smad1 can result in the
remodeling of the actin cytoskeleton (Aubin
et al., 2004). Thus, through the sequential use of overlapping
subsets of BMP ligands and receptors in a feed-forward mechanism, BMP
signalling could direct multiple stages in the development of a single class
of neurons.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/6/1119/DC1
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ACKNOWLEDGMENTS |
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