RESEARCH ARTICLE 1119
Development 135, 1119-1128 (2008) doi:10.1242/dev.012989
BMP type I receptor complexes have distinct activities
mediating cell fate and axon guidance decisions
Ken Yamauchi, Keith D. Phan and Samantha J. Butler*
The finding that morphogens, signalling molecules that specify cell identity, also act as axon guidance molecules has raised the
possibility that the mechanisms that establish neural cell fate are also used to assemble neuronal circuits. It remains unresolved,
however, how cells differentially transduce the cell fate specification and guidance activities of morphogens. To address this
question, we have examined the mechanism by which the Bone morphogenetic proteins (BMPs) guide commissural axons in the
developing spinal cord. In contrast to studies that have suggested that morphogens direct axon guidance decisions using noncanonical signal transduction factors, our results indicate that canonical components of the BMP signalling pathway, the type I BMP
receptors (BMPRs), are both necessary and sufficient to specify the fate of commissural neurons and guide their axonal projections.
However, whereas the induction of cell fate is a shared property of both type I BMPRs, axon guidance is chiefly mediated by only
one of the type I BMPRs, BMPRIB. Taken together, these results indicate that the diverse activities of BMP morphogens can be
accounted for by the differential use of distinct components of the canonical BMPR complex.
INTRODUCTION
Organisms develop using a remarkably small number of growth
factor families to specify a multitude of distinct cellular responses.
This biological economy is often achieved by feed-forward
mechanisms, where cells respond differentially to the same signal
received reiteratively over time. A striking example of this
phenomenon is the recent finding that molecules that act as
morphogens to induce the formation of diverse cell types in
developing tissues, also function as axon guidance cues in the
establishment of neuronal circuits (reviewed by Charron and
Tessier-Lavigne, 2005; 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
Department of Biological Sciences, University of Southern California, Los Angeles,
CA 90089, USA.
*Author for correspondence (e-mail: [email protected])
Accepted 9 January 2008
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 BMPmediated 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,
DEVELOPMENT
KEY WORDS: Axon guidance, Bone morphogenetic proteins, Commissural neurons, Morphogen, Spinal cord
1120 RESEARCH ARTICLE
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).
These results prompted us to determine whether the different
activities of the BMPs can be mediated solely through the canonical
BMPR complex by examining the contribution of the type I BMPRs,
BMPRIA and BMPRIB, to commissural axon guidance.
Biochemical studies have suggested that the type I BMPRs may
determine the specificity of BMP ligand binding (ten Dijke et al.,
1994); 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
Development 135 (6)
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 FITCcoupled 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-␮m
thick cryosections of E11.5 fresh-frozen mouse tissue as previously
described (Schaeren-Wiemers and Gerfin-Moser, 1993).
RESULTS
BMPRIB is expressed in postmitotic neurons in the
dorsal spinal cord
Previous studies have suggested that BMPRIA and BMPRIB are
expressed in the developing spinal cord (Dewulf et al., 1995; 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.
Expression constructs encoding constitutively active (ca) haemagglutinin
(HA)-tagged forms of both BMPRIA (human) and BMPRIB (mouse) were
described by Wieser et al. (Wieser et al., 1995) 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 ␮g/␮l plasmid DNA solutions, and electroporated and
processed as previously described (Briscoe et al., 2000). For the Math1
expression constructs, low concentrations (0.2 ␮g/␮l) 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).
Fig. 1. Distribution of type I BMPRs in the lumbar region of E11.5
mouse spinal cord. (A) BmprIa is expressed throughout the VZ and is
absent from the mantle layer, outlined in A and B. (B,C) BmprIb is
expressed specifically in the dorsal and intermediate VZ, and in the
dorsal-most neurons in the mantle layer (open arrowhead, C). The
yellow box in B is shown at higher magnification in C. (D) BmprIb is
expressed in an overlapping population of commissural progenitor cells
and neurons with that labeled by antibodies against Math1 (open
arrowheads, C,D). Scale bars: in A, 50 ␮m for A,B; in C, 25 ␮m for C,D.
DEVELOPMENT
Expression constructs and in ovo DNA electroporation
Distinct activities of type I BMPR complexes
RESEARCH ARTICLE 1121
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).
Consistent with previous reports (Timmer et al., 2002), 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).
DEVELOPMENT
Fig. 2. Dorsal neural cell identity is not
affected if either BMPRIA or BMPRIB are
constitutively activated in the chick spinal
cord after HH stage 14/15. (A-H) Following
electroporation of either CMV::caBMPRIA
(A,B,E,F,I,J) or CMV::caBMPRIB (C,D,G,H,K,L)
constructs in combination with a CMV::GFP vector,
changes in cellular identity were examined in stage
22/23 embryos using antibodies against (A-D)
pLh2 and (E-H) pIsl. There was no significant
difference in the number of dI1 and dI3 neurons
on the electroporated and non-electroporated
sides of the spinal cord (electroporated side
marked with +). (I-L) The activation status of the
BMP-specific Smad (Smad1/Smad5/Smad8) second
messenger intermediates was assessed using
antibodies against the phosphorylated (phos)
forms of Smad1/Smad5/Smad8, which
endogenously labels the progenitor domain of dI1
neurons (open arrowheads, I,K). Both constructs
can activate Smad1/Smad5/Smad8 to equally high
levels. (M) Quantification of the activity levels of
the CMV::caBMPRIA-HA or CMV::caBMPRIB-HA
constructs, by assessing the percentage of HA+
cells that were simultaneously positive for
phosSmad1/Smad5/Smad8. There was no
significant difference (P>0.17) between the
percentage of cells activated by BMPRIA
(97.0%±0.6 s.e.m., n=97 sections from 8
embryos) and that activated by BMPRIB
(96.0%±0.8 s.e.m., n=101 sections, 11 embryos).
(N) Summary of spinal cell types labeled.
(O) Quantification of the number of dI1 and dI3
neurons on the electroporated side verses the nonelectroporated side following electroporation of
either CMV::caBMPRIA or CMV::caBMPRIB at stage 11/12 or 14/15. For both receptors, there was a significant increase in the number of dI1
neurons (BMPRIA: P<0.0004, n=32 sections from 3 embryos; BMPRIB: P<3.8⫻10–5, n=32 sections, 2 embryos) and dI3 neurons (BMPRIA:
P<0.0032, n=25 sections from 2 embryos; BMPRIB: P<0.019, n=24 sections, 2 embryos) on the electroporated side, following electroporation at
stage 11/12. However, there was no increase in dI1 (BMPRIA: P>0.29, n=56 sections, 5 embryos; BMPRIB: P>0.48, n=36 sections, 4 embryos) or dI3
(BMPRIA: P>0.36, n=53 sections, 5 embryos; BMPRIB: P>0.22, n=35 sections, 6 embryos) cell numbers, following electroporation at stage 14/15.
Scale bar in L: 100 ␮m for A-L.
1122 RESEARCH ARTICLE
Development 135 (6)
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.
The constitutive activation of only BMPRIB results
in commissural axon guidance defects
To determine whether the type I BMPRs also transduce the BMP
axon guidance signal, we assessed the effect of constitutively
activating BMPRIA or BMPRIB (Akiyama et al., 1997) 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+
DEVELOPMENT
Fig. 3. BMPRIB specifically mediates commissural
axon outgrowth and guidance. (A-D) After the
ubiquitous expression of caBMPRIA and GFP from the
CMV enhancer, the (B) GFP+ (blue) and (C) Axonin1+
axons (green) cross the spinal cord normally at the FP
(arrowhead, B). (D) The electroporated Axonin1+ axons
also project around the circumference of the spinal
cord similar to control axons. (E-H) By contrast, after
misexpression of caBMPRIB and GFP from the CMV
enhancer, (F) no GFP+ axons cross the FP (arrowhead),
and (G,H) Axonin1+ axons are both mispolarized
medially towards the lumen of the spinal cord (arrows,
G,H) and stalled (open arrowhead) above the ventral
midline. + indicates the electroporated side. Scale bar
in A: 100 ␮m for A-H.
Distinct activities of type I BMPR complexes
RESEARCH ARTICLE 1123
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.
BMPRIB is required for commissural axon
reorientation
We also assessed the requirement for both BMPRIA and BMPRIB
by determining the consequence of functionally inactivating type I
BMPRs in mouse embryos. Single mutations in either of the type I
BMPRs have no effect on the fate of dorsal spinal neurons (WineLee et al., 2004). 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,
DEVELOPMENT
Fig. 4. Commissural axons are misguided
following misexpression of Math1::caBMPRIBIRES-fGFP in stage 15 chick spinal cords.
(A-D) Electroporation of either Math1::fGFP (A,B) or
Math1::caBMPRIA-IRES-fGFP (C,D) constructs results
in GFP (blue) in all commissural neural processes,
including the trailing processes (open arrowhead,
B,D) and Axonin1+ axons (green) that cross the FP
(arrowhead, B,D). Antibodies against Axonin1 also
transiently label MNs (m). (E,F) By contrast,
electroporation with a Math1::caBMPRIB-IRES-fGFP
construct results in commissural axons being
mispolarized towards the lumen (arrow, F) and
stalling (arrowhead, F). (G,H) The GFP+ axons in
control fillet preparations (G; dotted yellow lines in G
and H indicate position of the RP and FP) project
robustly to the FP, whereas GFP+ axons in the
Math1::caBMPRIB-IRES-fGFP fillets (H) do not enter
the ventral spinal cord (arrowhead). (I,J) The extent of
the commissural axon outgrowth was quantified in
stage 22/23 embryos by determining the number of
GFP+ commissural axons that crossed lines drawn (J)
in the mid-dorsal (MD), intermediate (INT) and midventral (MV) spinal cord, and the FP. Of the control
commissural neurons extending axons to the MD line,
over 55% of these axons subsequently project to the
MV line (n=158 sections from 10 embryos). By
contrast, less than 23% of the Math1::caBMPRIBIRES-fGFP commissural axons that extend to the MD
line subsequently reach the MV line (n=145 sections,
15 embryos), a figure significantly different from
control (P<2.4⫻10–31). Scale bar in F: 100 ␮m for A-F.
Fig. 5. Commissural axons are mispolarized in BmprIb loss-offunction mutant mice. (A-C) In transverse sections of the spinal cord
taken from hind-limb levels from E11.5 wild-type (A) and Math1::cre;
BmprIaflox/flox (B) embryos, Tag1+ neurons are highly polarized, with the
overwhelming majority (99.9%±0.1 s.e.m., n=27 sections from 4
embryos) extending axons to the FP. Many commissural axons also
extend towards the FP in BmprIb–/– embryos (C); however, a small
number of axons (1.8%±0.4 s.e.m., n=50 sections, 5 embryos) project
aberrantly towards the lumen (open arrowhead, C). (D) A significantly
higher (P<0.001) percentage of commissural axons are mispolarized
medially in BmprIb–/– embryos compared with their wild-type
littermates. (E,F) The Math1::cre line drives expression of Cre
recombinase (green) specifically in the pLh2+ (red) population of
commissural neurons. Scale bar in A: 75 ␮m for A-C, E,F.
2003). This result suggests that the loss of BMPRIB, but not
BMPRIA, results in a perturbation of the commissural axon
trajectory in vivo.
To examine the response of wild type, BMPRIA- and BMPRIBdeficient commissural axons to the repellent activity of the RP,
whole-mount fillet preparations of the spinal cord were taken from
E11.5 mouse embryos. In fillet preparations, the spinal cord is
opened ventrally like a book making it possible to examine the
trajectory of the commissural axons immediately adjacent to the RP.
Both wild-type (open arrowhead, Fig. 6A) (Butler and Dodd, 2003)
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–/–
Development 135 (6)
Fig. 6. Commissural axons are dorsally mispolarized in vivo in
BmprIb–/– single mutants and Math1::cre;BmprIaflox/flox; BmprIb–/–
double mutants. (A,B) In either wild-type (A) or BMPRIA-deficient (B)
fillets of the spinal cord, very few Tag1+ axons extend towards the RP
(A, open arrowhead) and no axons cross the RP. (C-F⬘) By contrast,
many Tag1+ axons extend into the RP in either BmprIb–/– single mutants
or Math1::cre;BmprIaflox/flox; BmprIb–/– double mutants (open
arrowheads, C,F), with commissural axons (closed arrowheads, D’,F’)
now observed to cross the RP (outlined in D,F). (G) There is no
significant difference (P>0.27) between the percentage of mispolarized
axons in BmprIaflox/flox control fillets (0.75%±0.19 s.e.m., n=8213 pLh2+
neurons from 9 embryos) and the BMPRIA-deficient
(Math1::cre;BmprIaflox/flox) fillets (0.95%±0.20 s.e.m., n=9262 pLh2+
neurons, 9 embryos). By contrast, a significant increase (P<0.002) is
observed in BmprIb–/– mutants (1.32%±0.14 s.e.m., n=7940 pLh2+
neurons from 10 embryos) compared with wild-type litter-mates
(0.73%±0.10 s.e.m., n=10379 pLh2+ neurons, 12 embryos). The
percentage of mispolarized commissural axons seen in fillets from the
Math1::cre;BmprIaflox/flox; BmprIb–/– double mutant embryos
(3.5%±0.35 s.e.m., n=2114 pLh2+ neurons, 2 embryos) is statistically
identical (P>0.4) to that seen in fillets from Gdf7–/– embryos (Butler and
Dodd, 2003). Scale bar in B: 10 ␮m for A-F⬘.
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
DEVELOPMENT
1124 RESEARCH ARTICLE
Distinct activities of type I BMPR complexes
RESEARCH ARTICLE 1125
Fig. 7. In the absence of BMPRIB, mouse
commissural axons are significantly less
responsive to a rat RP explant. (A,A’) E11.5
rat RP explants deflect Tag1+ commissural axons
(green) growing within E10.5 wild-type mouse
dorsal spinal cord (d-sc) explants by an average
angle of reorientation of 21.8°±2.0 s.e.m.
(n=28). (B,B’) Commissural axons deficient in
BMPRIA (Math1::cre;BmprIaflox/flox) are reoriented
by a rat RP explant, to a similar extent (P>0.4) as
BmprIaflox/flox control littermates, with an average
reorientation angle of 20.4°±2.0 s.e.m. (n=18).
(C,C’) By contrast, BmprIb–/– commissural axons
are significantly less reoriented by a rat RP
explant than wild-type littermates (P<1.3⫻10–7),
with an average angle of reorientation of
9.6°±1.9 s.e.m. (n=19). Note that in the tracings
in A’, B’ and C’, only the axons deriving from the
mouse d-sc explant are illustrated. (D) Schematic
to illustrate the orientation of the E11 rat RP
explant and the E10.5 mouse d-sc explant.
(E) Histogram of the average angles of
reorientation. Scale bar in C: 75 ␮m for A-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
The discovery that inductive growth factors, such as the BMPs, have
dual activities at different times in development, acting as both
morphogens and axon guidance cues, has suggested a model in
which the signals that initially establish the cellular fate of neurons
are subsequently reused to specify the pattern of axonal trajectories.
However, it remains unclear how these growth factors result in such
different cellular outcomes during development. To address this
question for the BMPs, we have determined that one of the canonical
type I receptors, BMPRIB, is both necessary and sufficient to
mediate the known guidance activities of the RP chemorepellent.
Thus, the feed-forward mechanism that underlies the ability of the
BMPs to signal different activities to developing commissural
neurons does not depend on divergent receptor signalling, as had
been seen for other morphogens, rather it requires the sequential use
of the canonical BMPR complex. However, the type I BMPRs do
not function interchangeably in this process, rather the exact
composition of the canonical BMPR complex crucially determines
the nature of the response of commissural neurons to the BMP signal
(Fig. 8).
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
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.
The phenotype of the BmprIa; BmprIb double mutants suggests
that BMPRIA might have weak activity as an axon guidance
receptor. However, BmprIa is not present in postmitotic
commissural neurons (Fig. 1) and our gain-of-function studies (Figs
3, 4) suggest that the misexpression of BMPRIB, but not BMPRIA,
affects commissural axon outgrowth and guidance. These
observations are more consistent with a model in which BMPRIA
redundantly contributes to the establishment of neuronal polarity
through an earlier role in the specification of commissural cell fate,
rather than BMPRIA acting directly to mediate axon guidance. Since
it is difficult to separate a polarizing activity from a guidance activity
in vivo, we used the in vitro reorientation assay to further assess the
response of wild-type, BMPRIA and BMPRIB-deficient
commissural axons to the RP chemorepellent. The reorientation
assay is a robust and sensitive measure of guidance activity
(Augsburger et al., 1999; 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⬘).
1126 RESEARCH ARTICLE
Development 135 (6)
Fig. 8. BMPs have diverse functions for progenitor and
postmitotic neurons in the dorsal spinal cord during
development. (A) Graded signalling from the RP-derived BMPs is
sufficient to induce the dI1, dI2 and dI3 cell fates. BMPRIA and BMPRIB
have a shared redundant activity mediating dorsal neural cell fate
specification, presumably acting through the Smad transcriptional
regulator. (B) Subsequently, BMP heterodimers act as a diffusible
chemorepellent to direct (dI1) commissural axons away from the RP.
This activity is predominantly mediated by BMPRIB, which acts through
an as yet unknown second messenger intermediate to locally
reorganize the cytoskeleton.
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.
Of the type I BMPRs, BMPRIB is primarily responsible for
translating the gradient of BMPs from the RP into axon guidance
cues for commissural neurons. BmprIb is specifically expressed in
postmitotic dorsal neurons and introducing constitutively active
forms of BMPRIB, but not BMPRIA, into the developing spinal
cord results in the misprojection of axons into the VZ. The extent to
which the direction of outgrowth was randomized remains unclear,
since it was not possible to assess whether electroporated axons were
mispolarized dorsally. Farnesylated GFP fills the entire neuronal
process making it difficult to distinguish dorsally projecting axons
from trailing processes. Additionally, more severe axon guidance
defects might have been observed had it been possible to use a form
of caBMPRIB that was completely independent of ligand activation.
For both type I caBMPRs, although the activation of the receptor no
longer requires ligand binding, the activity of the caBMPRs can be
further enhanced by ligand binding (Akiyama et al., 1997). 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
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-offunction 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
DEVELOPMENT
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.
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.
We are most grateful to Ester Stoekli for Axonin1 antibodies; Anthony Celeste
and John Wozney for the type I BMPR in situ constructs; Jay Timmer, Lee
Niswander and Kohei Miyazono for the type I caBMPR constructs; Karen Lyons,
Sean Brugger and Robert Pogue for the type I BMPR deficient mice; and David
Rowitch and Sovann Kaing for the Math1::cre transgenic line. We would also
like to thank James Briscoe, Jane Dodd, Virginia Hazen, Artur Kania, Jane
Johnson, Ben Novitch, Michael Quick and Peter Schieffele for helpful
discussions; and James Briscoe, Ben Novitch, Jeanette Perron and Jonah Chan
for comments on the manuscript. This work was supported by grants from the
James H. Zumberge Research and Innovation Fund, and the March of Dimes
Foundation.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/6/1119/DC1
RESEARCH ARTICLE 1127
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1128 RESEARCH ARTICLE