Expression of stabilized β-Catenin in the skin leads to inappropriate Wnt6 expression
To investigate the effect of meningeal secretory molecules on embryonic brain development, we set out to generate a transgenic mouse line with expanded meninges using a meninges-specific cre mouse line. We decided to try using an Msx2
-Cre mouse line which uses the 439bp 5′ flanking region of the mouse Msx2
gene and crossed the line with exon3 floxed β-Catenin (Ctnnb1lox(ex3)
, shortened as bE3
in the figures) to express stabilized constitutively active β-catenin in the meninges (Harada et al., 1999
; Sun et al., 2000
). The native Msx2
gene is normally expressed in the meninges (Rice et al., 2003
), however, the transgenic Msx2
-Cre line drives recombination not in the meninges but instead in the skin, revealed by crossing with the ROSA-YFP CRE reporter line ( and low magnification in Suppl. Fig. 1B
) and the Rosa-LacZ CRE reporter line (Suppl. Fig. 1C
). There was no YFP or LacZ expression in the meninges or any regions of the brain. Msx2
-Cre drove recombination starting from E13.5 and the YFP expression persisted postnatally (Suppl. Fig. 1
and data not shown). At E16.5, the Msx2
mutant embryos were smaller and the skin over the head was thinned and the skull malformed (). Persistent activation of the Wnt pathway in the skin of the Msx2
mutants led to increased expression of Axin2
, a well-established Wnt target gene (Jho et al., 2002
), in the skin (). Interestingly, there was also increased expression of the Wnt responsive transcription factor Lef1 in a non-autonomous manner in the underlying mesenchymal tissue, including the meninges (). This made us wonder if there was Wnt signaling dependent expression of a Wnt ligand in the skin that was then signaling to the underlying tissues. Wnt6
is normally expressed high laterally and low dorsally in the skin of the head () and is absent from the midline. However, in the Msx2
expression covered the entire dorsal surface of the head. We also found that Wnt6 was elevated about 1.5 fold compared to control using quantitative real time PCR (qPCR) on mRNA isolated from whole head (Suppl. Fig. 2C
). Expression of Wnt10b
, another skin-specific Wnt, was not altered () indicating that persistent activation of the Wnt signaling pathway in the skin leads to specific upregulation of Wnt6 expression.
Figure 1 Msx2-Cre drives recombination of Ctnnb1lox(ex3) in the skin and leads to ectopic Wnt6 expression and callosal agenesis. A-A″) The ROSA-YFP reporter reveals the recombination pattern for Msx2-Cre in the outermost layer of the head, the skin, which (more ...)
Mutants with persistent activation of the Wnt pathway in the skin are acallosal
Beyond the skin and calvarial defects, we found that in the Msx2
mice the main cortical commissural pathway, the corpus callosum, failed to form (). However, in the same mutants other commissural pathways including the anterior commissure and hippocampal commissures were still formed (although the hippocampal commissure is slightly smaller in size than normal) (). At E17.5, a day before the mutant embryos die, it was apparent that the callosal axons stopped at the cortical midline and rather than crossing formed Probst bundles (* in ), which are aberrant axonal tracts made up of callosal axons that fail to cross the midline (Paul et al., 2007
). These callosal defects were also observed in horizontal sections of mutant animals (Suppl. Figure 1A
) and showed full penetrance from fourteen mutant embryos analyzed. Since the failure of corpus callosum formation is a dramatic midline structural defect, we wondered how excess Wnt signaling in the dorsal skin might cause this phenotype. There have also been numerous studies showing strain differences in the appearance of corpus callosum defects in mice with some strains (eg 129 and Balb/c), however, our colonies of Msx2
-Cre and Ctnnb1lox(ex3)
mice have been extensively crossed into the CD-1 background, not noted for defects in the corpus callosum.
One possible cause of agenesis of the corpus callosum could be defects in the development of the cortical projection neurons. This phenotype has been often observed in mutant animals for the transcription factors governing maturation of the cortical callosal neurons comprising layer II/III (Alcamo et al., 2008
; Armentano et al., 2006
; Britanova et al., 2008
; Molyneaux et al., 2007
; Paul et al., 2007
; Piper et al., 2009
; Shu et al., 2003
). We examined the expression of several of these factors, such as Satb2
, and found that their expression was intact in the Msx2
brains at E15.5, a day before the formation of the corpus callosum (Suppl. Figure 1D
). Some of the earlier born neurons that make up layer V/VI also contribute axons to the corpus callosum, so we also examined Ctip2 and Tbr1, two markers of these early born neurons. We found that the laminar organization of the mutant cortex was similar to wild type littermates. We also did not see any changes of the proliferative zone using an M-phase cell cycle marker (phospho-histone H3 - pH3), a ventricular zone progenitor markers (Nestin and Pax6) or a marker for the basal intermediate progenitors in the subventricular zone (Tbr2) (Suppl. Fig. 2A
). Another potential cause of callosal agenesis in these mice may be alterations in expression of guidance molecules, such as semaphorins, slits, Wnt5a, Draxin and ephrins, expressed in the cortical midline and previously shown to regulate callosal axonal crossing (Bagri et al., 2002
; Islam et al., 2009
; Keeble et al., 2006
; O’Donnell et al., 2009
; Paul et al., 2007
). To address this we examined expression of a panel of these ligands and their receptors in our mutant mice but did not observe any obvious differences in the pattern of expression between mutant and control brains (Suppl. Fig. 2B
Wnt6 induces expansion of neural crest derived meningeal tissues
We wondered whether the excess Wnt6 in the head itself might be an inhibitor of corpus callosum formation, so we electroporated Wnt6 into the cortical midline prior to callosum formation and found that the corpus callosum still formed normally (data not shown). We reasoned that another possible mechanism for callosal agenesis might be via the known role of Wnts as a growth factor for neural crest cells. Since the meninges overlying the cortex originate from the cranial neural crest (Serbedzija et al., 1992
) and Wnt6 induces expansion of cranial neural crest cells in avian species (Garcia-Castro et al., 2002
; Schmidt et al., 2007
), we looked for meningeal abnormalities in the Msx2
mutants. We examined meningeal development at E14.5-E15.5, before the formation of the corpus callosum in control and mutant mice. Using Ki-67, a cell proliferation marker, we found that meningeal cell proliferation was elevated in Msx2
mutants () and this is consistent with our findings of ectopic Axin2 and Lef1 expression (). Furthermore, using an anti-Zic1 antibody, which labels meningeal cells (Inoue et al., 2008
), we found expanded meninges both over the surface of the cortex, and, even more interestingly, in the interhemispheric fissure where the corpus callosal axons will eventually form (). To more carefully examine the three meningeal layers, we used markers specific for each layer expressed during embryonic development (Siegenthaler et al., 2009
; Zarbalis et al., 2007
normally is expressed in the developing arachnoid meninges, but in the mutants it was ectopically expressed under the skin implying that Wnt6-induced meningeal progenitors to produce extra arachnoid cells (). We also used anti-Foxc2 antibody which labels predominantly the dural meningeal precursors (Zarbalis et al., 2007
); Foxc2 expressing dural cells are also expanded in the mutant embryos (). Cxcl12, which labels the inner pial layer, was likewise expanded and ectopically expressed in the mutants (). These results suggest that ectodermal Wnt6 induces pia, arachnoid and dural meningeal cells to overgrow both over the surface of the cortex but also between the hemispheres. We suspected that the non-cell autonomously expanded meninges may be the cause of the corpus callosal agenesis and that perhaps we could use the Msx2
mutants to identify a normal role for the meninges in corpus callosum development.
Figure 2 Meningeal expansion by activation of Wnt6 in the skin and increased expression of BMP7. Low-magnification (A) and high-magnification (A′ and B) of Zic immunostaining in WT and Msx2-Cre; Ctnnb1lox(ex3) E14.5 heads. Lateral (A′) and medial (more ...)
Elevated Bmp signaling in the meninges and cortex in mutant mice
expression zone marks the areas of epithelial-mesenchymal transition and Bmp signaling is known to be involved in this transition (Kalluri and Weinberg, 2009
; Lavery et al., 2008
), and in the mutant embryos, the development of the skull vault appeared compromised (). Since the normal source of BMPs for much of skull development is the meninges (Kim et al., 1998
; Rice et al., 2005
), we hypothesized that the ectopic meningeal tissue may express BMP signaling components affecting the skeletogenic mesenchyme for the mouse skull vault. This also raised the possibility that excess Bmps produced by the meninges might interfere with corpus callosum development. We examined the expression of several Bmp ligands and found that BMP7
was ectopically expressed in the excess meningeal tissue at E14.5-15.5 and that the overall expression level in the meninges was increased (; Suppl. Figure 2C
). To determine whether the increased expression of BMP7
alters the underlying Bmp signaling levels within the cortex, we stained the brain with an anti-pSMAD1/5/8 antibody. This showed that the mutant brains had increased phosphorylation of SMAD1/5/8, confirming that the ectopic BMP7
expression in the meninges is biologically active and increases cortical Bmp signaling (). Interestingly, the increased phospho-SMAD staining was barely present at E13.5 but dramatically increased by E15.5 (), in parallel with the onset of recombination in the skin. Phosphorylation of SMAD2, which is a downstream target of canonical TGF-β ligands rather than Bmp ligands, was not induced (data not shown). An interesting supporting point for the increased Bmp signaling in the developing cortex that we see with these markers is increased Zic1 expression in the cortical ventricular zone (). Zics are known to be induced by Bmp ligands in the developing CNS (Aruga et al., 2002
Mice with excess meninges have generally normal midline glial development and appropriate production of cingulate pioneer neurons
There has been extensive study of the development of the corpus callosum and it’s clear that some of the causes of callosal agenesis are due to structural defects at the cortical midline leading to failure of the fusion of the midline (Paul et al., 2007
). We wanted to determine whether the excess meningeal tissue or the increased BMP signaling leads to defects in the structural elements required for corpus callosum formation. We examined whether the midline glial structures critical for development of the corpus callosum (Shu and Richards, 2001
; Smith et al., 2006
) were affected in the mutants. Staining the E16.5 cortex with BLBP and GFAP, markers for glial wedge cells, did not show alterations (). We also used BLBP to examine the glial wedge both before (E15.5) and after (E17.5) the callosum forms and found that before the callosum forms there is no clear defect in the organization of this structure (). The glial wedge is made up of radial glial cells that function both as progenitor cells for the Calretinin-expressing medial cingulate neurons that produce the pioneer axons crossing the callosum and as part of the scaffolding for these crossing axons (Rash and Richards, 2001
). Anti-Calretinin antibody labeled these cingulate neurons and their axons () and the mutants did not show any difference in the numbers of these cingulate cortical neurons although there were apparent defects in their axon projections across the midline (). We also dated the birth of Calretinin+ neurons in the cingulate cortex with BrdU injections and found that Calretinin+ neurons were born around E12.5 (data not shown). Staining for BrdU at E14.5 also did not show any apparent difference in the number of neurons born at E12.5. In addition, the subcallosal sling is made up of a group of migratory neurons, frequently visualized with NeuN staining (Shu and Richards, 2001
), that make up the ventral limit of callosal axon projection. In both control and experimental acallosal mice, NeuN clearly labeled this group of glial sling neurons (). All of these results suggest that neither a mechanical effect of the excess meninges nor the ectopic BMP7
expressed by the meninges affected the cingulate pathfinding neuron generation, the midline glial wedge or the glial sling neurons in the mutant medial cortex but that nevertheless the axons failed to cross the midline.
Figure 3 A) Midline glial structures are not affected in the mutant but pathfinding axons fail to cross the midline. Immunostaining of BLBP and GFAP shows midline glial structures and NeuN shows subsling migratory neurons in the WT and Msx2-Cre; Ctnnb1lox(ex3) (more ...)
Midline BMP7 inhibits callosal axon outgrowth
Although the Calretinin+ cingulate neurons were generated, their axons failed to cross the midline in the mutant cortex (). Given that the glial wedge and sling are apparently minimally affected, we hypothesized that increased BMP7 released by the meninges might affect axon outgrowth of cingulate cortical neurons. To test this we introduced a BMP7 expression construct into the E13.5 embryonic cortical midline by in utero electroporation. We examined the electroporated brains at E15.5, a day before the initial pioneer axons should cross the midline. BLBP+ midline glial structures appeared normal, consistent with the previous results obtained from mutant brains, however, Calretinin+ cingulate cortical axons were disorganized in the electroporated hemisphere (). By E17.5, when the corpus callosum should have formed, we found that BMP7 had potently inhibited formation of the corpus callosum (). This effect was specific for BMP7 since BMP6 expression in the same region did not affect callosum formation (). Considering the disorganization of pioneer axons at E15.5 in the midline by BMP7 overexpression, this suggests that BMP7 protein acts as an inhibitor of pioneer callosal axon outgrowth, although another possibility is that excess BMP7 in the cortex leads to abnormalities in the meninges at the midline.
Figure 4 Failure of callosal axon growth caused by BMP7 overexpression. Electroporation of Bmps (A and B) and Bmp receptors (C) into the E13.5 medial cortex. A) Immunostaining of BLBP and Calretinin in BMP7 electroporated cortex. The BLBP and Calretinin staining (more ...)
To address this latter question we used a cell autonomous means to mimic the activation of Bmp signaling in the cingulate cortical neurons by expressing a constitutively active form of type I Bmp receptor (CA-Bmpr1a) in the medial cortex from E13.5 to E16.5, when the first cingulate callosal axons cross the midline. We compared this to eGFP controls as well as overexpression of dominant negative forms (DN-Bmpr1a) (). This experiment showed that cell-autonomous activation of BMP signaling in the cingulate cortical neurons inhibited the growth of corpus callosal axons in the electroporated hemisphere, however, the dominant-negative form of type I Bmp receptors had no apparent effect on callosum formation (). This result supports the idea that BMP7 in the midline meninges acts as an inhibitor for corpus callosal axon crossing the midline and rules out the possibility that BMP7 expressed within the cortex is non-autonomously acting on meningeal cells and reciprocally inhibiting callosal outgrowth. One of the important features of these experiments is that manipulation of one side of the cortex apparently is sufficient to block the formation of this commissure with bilateral contributions. This suggests that the initial formation of the callosum by Calretinin+ cingulate pioneer neurons involves interaction of these axons from both sides at the midline, perhaps via a mutual handshake.
Mice with mid-corticogenesis meningeal defects have expansion of the corpus callosum
Our initial observations are consistent with the idea that BMP7, expressed by the meninges, is a potent negative regulator of corpus callosum formation. Our data mostly rests on the generation of a novel mouse mutant that has meningeal overgrowth, although the direct ectopic expression of BMP7 within the cortex also blocks callosum formation. To strengthen our arguments we wanted to develop comparable loss-of-meningeal function mouse mutants that might allow us to confirm the negative role of the meninges in the formation of the callosum. We wished to undertake two approaches toward this goal but first needed to identify a meninges selective Cre line. Preferably, one that begins expression in the meninges at a later developmental timepoint thus allowing us to generate mice with a more limited meningeal phenotype. To this end, we tested the Pdgfr
β-Cre line (Foo et al., 2006
), and found that it is active in some meningeal derivatives in addition to blood vessel associated pericytes (also neural crest-derived cells) beginning at about E13.5 ().
Figure 5 Enlarged corpus callosum in the loss of function meninges mutants. A) ROSA-LacZ CRE reporter line shows Pdgfrβ-Cre expression in the midline meninges at E14.5. LacZ is also expressed in the pericytes associated with blood vessels widely in the (more ...)
We previously showed that Foxc1
mutant mice have major defects in meningeal development (Siegenthaler et al., 2009
; Zarbalis et al., 2007
) and that these mice largely lack meningeal cells over much of their cortex, including the medial cortex. Failure to form normal meninges leads to detachment of the radial glial cells from the basement membrane and also major neurogenic defects (thus lacking most callosal neurons) (Siegenthaler et al., 2009
; Zarbalis et al., 2007
). However, using the Pdgfrβ
-Cre line and a conditional Foxc1lox
line we generated mice with a later deletion of Foxc1 that have a relatively intact brain organization. Analysis of these late meningeal Foxc1
mutants at E15.5 shows that there is reduced meningeal BMP7 expression in these mice both over the cortex and in the interhemispheric fissure (). Zic1+ meningeal cells are diminished in the interhemispheric fissure of Pdgfrβ
mice, indicating that decrease in BMP7 is likely due to a reduction in BMP7-expressing meningeal cells (, we used ‘fl’ for floxed allele in the figures).
mutant mice have excess meninges due to increased production of Wnt6 by the overlying skin. Expansion of the meninges is accompanied by increased expression of a target of the Wnt signaling pathway (Axin2) as well as a Wnt signaling mediator (Lef1). This suggests that canonical Wnt signaling may be an important component of meningeal development. Indeed, previous studies using the Wnt1-Cre line crossed with the Ctnnb1lox(lof)
allele had shown a failure of formation of many cranial neural crest components (Brault et al., 2001
), however, this phenotype is developmentally too early to evaluate callosal crossing. Instead, we crossed the Ctnnb1lox(lof)
with the Pdgfrβ
-Cre line and found that, similar to the Pdgfrβ
mutants, there was a notable decrease in meningeal BMP7 and a reduction in interhemispheric meningeal cell numbers ().
We next used our two novel meningeal mutants to determine how loss of midline BMP7 affects callosal crossing. In addition to the reduced expression of BMP7 in the meninges () there were markedly decreased levels of phospho-SMAD1/5/8 activity in the medial cortex of both mutants (). Thus, these mice apparently have the opposite phenotype of the Msx2-Cre;Ctnnb1lox(ex3) mice in that they have less interhemispheric meninges, and, consequently, reduced BMP7 and BMP signaling. Next, we examined the development of the corpus callosum in these mice and found that remarkably, the Pdgfrβ-Cre;Ctnnb1lox(lof) and Pdgfrβ-Cre;Foxc1lox mice had larger corpus callosums than their littermate controls with more axonal fibers crossing (). We quantified this difference by measuring the corpus callosum in cohorts of mutants and control mice and found that the callosum was significantly increased in size at E17.5 throughout the rostro-caudal anatomic levels of the corpus callosum ().
One important question to address is why the callosal size is increased in these mice. One possibility is that the callosum is larger because it begins to be formed earlier (due to loss of Bmp7 from the meninges) and thus is larger at the stages we examined. To address this, we examined the size of the callosum in these two mutant lines at an earlier stage when the callosum has just started forming, E16. At this time the mutant mice still have a marked increase in callosal size (), consistent with the idea that the callosum begins to form early in these mutants and is thus at a more advanced stage of development at E17.5.
Another potential mechanism for increased callosal size is that there could be an increased number of neuron types that contribute to the callosum in these mutant lines due to potential effects of the meninges on cortical development. To address this we examined the expression of layer specific markers in the developing cortex of both lines as well as the Msx2
mice. Interestingly, we found that the Pdgfrβ
but not the Pdgfrβ
mice have an alteration in the numbers and distribution of superficial neurons that would contribute to the callosum (Supplemental Figure 3
). Since we observed this phenotype in only one of the lines, we suspect that this is not the cause of the increased callosal size, rather it is accelerated formation of the callosum due to early crossing in mice lacking Bmp7 at the midline.
Wnts are positive regulators of callosal axon growth
Our data thus far is consistent with the idea that the meninges normally limit the formation of the corpus callosum and that one of the important mediators of this function is BMP7 expressed by the meninges acting on the medial cortex and cingulate pathfinding axons. One puzzling aspect of these observations is the fact that normally BMP7
is expressed in the midline meninges, albeit at lower levels, yet these axons still do manage to cross the midline in the face of the normal presence of BMP7. Why does the callosum ever form if BMP7 is always present in the meninges? The corpus callosum is the only cortical structure where axons make trajectories across meningeal tissues. In this sense, it seems possible that there is a BMP7-counteracting molecule in the cortical midline that is induced prior to formation of the corpus callosum and that the action of BMP7 produced by the meninges is in part to prevent premature formation of the corpus callosum until this positive influence is produced. Since Frizzled-3
mutant mice, which fail to transduce much Wnt signaling in the cortical projection neurons, also fail to form the corpus callosum (Wang et al., 2002
) and Wnt signaling is critical for axon guidance in other areas of the nervous system (Agalliu et al., 2009
; Bovolenta et al., 2006
; Ciani and Salinas, 2005
; Dickson, 2005
; Krylova et al., 2002
; Lyuksyutova et al., 2003
; Maro et al., 2009
; Schmitt et al., 2006
), we wondered whether a Wnt signal might be the positive cue allowing pioneer axons to cross the midline. To address this, we inhibited endogenous Wnt signaling in the medial cortex of normal mice by electroporating the soluble Wnt inhibitor Dkk1 into the cortical midline. This resulted in the failure of cingulate axons to cross the midline and led to callosal agenesis (), indicating a probable role for Wnt signaling in this process and making a Wnt ligand a possible key regulator of the formation of the callosum.
Figure 6 Wnt3 antagonizes the negative effects of BMP7. A) Wnt signaling is required for corpus callosum formation. Immunostaining of GFP and Calretinin in E16.5 brains electroporated with eGFP or Dkk1 at E13.5 shows that inhibiting Wnt signaling at the midline (more ...)
In order to determine the likely endogenous Wnt ligand responsible for this function, we screened expression of Wnt genes by in situ hybridization to see which Wnt is expressed in a manner supporting a role in cingulate cortical axon projection toward the contralateral cortex. We found that Wnt3 is expressed in a subpopulation of the midline cingulate cortical neurons in control mice at E14.5, just before the callosum is formed (). Interestingly, in the Msx2-Cre;Ctnnb1lox(ex3) embryos at E14.5, Wnt3 expression was absent where the corpus callosum should appear in 1.5 days (). Thus, Wnt3 is apparently expressed in the right location to be the Wnt ligand regulating callosum formation and strikingly, is also missing from acallosal Msx2-Cre;Ctnnb1lox(ex3) mutant mice.
Wnt3 antagonizes the negative effects of BMP7 on callosal axon outgrowth and rescues the callosum of mutant mice
Our results suggest a model that Wnt3 is expressed in cingulate neurons at the cortical midline in order to act locally to oppose the negative influence of BMP7 from the meninges and that the appearance of Wnt3 at E14.5 is a crucial step toward corpus callosum formation. We designed both in vivo
and in vitro
experiments to test whether Wnt3 interacts with BMP7 in regulating callosal axon growth. Initially we tested whether the meninges produce a secreted chemorepellent using collagen explant assays. We embedded explants of cingulate cortex in collagen and confronted them with either meningeal explants from control and mutant mice with excess meninges (the Msx2
mice) or COS7 aggregates expressing BMP7
. These experiments revealed no evidence of any repellent effect at a distance by the meninges (Supplemental Figure 4
). Next, in order to determine if there is a more short-range or contact dependent effect, we collected midline cortical neurons from E14.5 embryonic brains and co-cultured cortical neurons with COS7 cells expressing BMP7
or both ligands. In these cultures, calretinin+ axons frequently grew out but failed to grow across BMP7 expressing COS7 cells, similar to the effects of many axonal growth inhibitors in other systems (Law et al., 2008
; Niederkofler et al., 2010
). To quantify this effect we counted the number of axons that approached BMP7-expressing COS7 cells but didn’t cross them (Type A axons) and axons that grew across the BMP7-expressing COS7 cells (Type B axons) and found that BMP7 is a potent inhibitor in this assay, essentially causing 100% of the cells to be Type A, compared to GFP transfected COS7 cells (). Wnt3
expression alone had no clear effect on axon growth compared to control. In both the Wnt3 and GFP conditions many axons (about 60%) freely grew across the COS7 cells (). To test the ability of Wnt3 to antagonize the negative effects of BMP7 in this assay we co-expressed the ligands and found that Calretinin+ axons now quite readily crossed BMP7+Wnt3-expressing COS7 cells (, p<0.001). Thus, Wnt3 apparently has minimal if any stimulatory effect on axon growth in this assay, unless BMP7 is present, in which case it apparently counteracts the negative effects of BMP7.
To examine this interaction in vivo, we introduced BMP7 along with Wnt3 in utero. Strikingly, we observed formation of the corpus callosum when we expressed Wnt3 expression along with BMP7 (). Thus, it appears that Wnt3 is able to counteract the negative effects of BMP7 on callosal pathfinding axon outgrowth. This is consistent with the onset and spatial distribution of Wnt3 at E14.5 being a critical regulator of callosum formation by allowing the pioneer axons to cross the BMP7 expressing midline meninges.
Figure 7 Co-expression of Wnt3 blocks the negative effects of BMP7 in vivo. A) Co-electroporation was conducted with the combination of BMP7 with eGFP or Wnt3 at E13.5 and the brains were analyzed at E17.5 for callosal agenesis. B) Wnt3 overexpression rescues (more ...)
Since the mutant cortex lost Wnt3 expression before the initial pioneer axons crossed the midline, we wondered whether adding back Wnt3 would rescue the failure of the pioneer axons crossing the midline in the mutants with excess meninges (the Msx2-Cre;Ctnnb1lox(ex3) mice). To test this, we electroporated a Wnt3 expression construct into the midline cortex of Msx2-Cre;Ctnnb1lox(ex3) mice at E13.5 and examined E17.5 embryos and found that TAG1 and L1 positive corpus callosal axons are obvious in the Wnt3 electroporated brain but GFP-electroporated brains failed to form the midline callosal trajectories ().
To further address our hypothesis that Wnt3 signaling antagonizes BMP7 signaling allowing the corpus callosal axons to cross the midline, we examined staining for pSMAD1/5/8 in the medial cortex of BMP7 electroporated mice either with GFP or Wnt3 co-electroporation. In mice that were electroporated with BMP7 and GFP, as expected, the level of pSMAD1/5/8 immunoreactivity was markedly increased in the BMP7 electroporated medial cortex (). However, when Wnt3 was co-electroporated with BMP7 and the brains examined three days later, the pSMAD1/5/8/levels were blunted and perhaps even lower than seen in the opposite unelectroporated hemisphere (). To quantify these effects we performed western blotting for pSMAD1/5/8 and compared it to the signal using an antibody that sees all SMAD1 or GAPDH to normalize. In these experiments we found that BMP7+eGFP electroporated cortex had a 40% higher level of pSMAD1/5/8 compared to cortex electroporated with Wnt3+BMP7 (Supplemental Figure 5A–B
). We also examined the fluorescence intensity ratio of the tissues labeled with pSMAD1/5/8 antibody on the electroporated side compared to the opposite hemisphere in BMP7+eGFP electroporated mice vs Wnt3+BMP7 electroporated mice (essentially quantitating the signal in but in five new electroporated embryos in each condition) and found a dramatic difference in the pSMAD1/5/8 ratio between the two conditions (Supplemental Figure 5C
Gdf5 from Cajal-Retzius Cells and its inhibitor Dan from the meninges control Wnt3 expression and regulate corpus callosum formation
Our analysis so far has led us to propose that BMP7 helps to block the formation of the corpus callosum by inhibiting callosal pioneer axon outgrowth, thereby inhibiting formation of the corpus callosum until such time as Wnt3 expression begins in the pioneer neurons and antagonizes the effects of BMP7, allowing initial callosal axon outgrowth. This left us with one significant puzzle – why were the Msx2
mice missing the expression of Wnt3? We hypothesized that another secreted factor normally produced by the meninges was also overexpressed in the mutants and that this factor helps to regulate Wnt3 expression in the cingulate cortex. Our thoughts immediately turned to the Gdf5/6/7 inhibitory molecule Dan (Dionne et al., 2001
), which some time ago we showed is expressed by the meninges (Kim and Pleasure, 2003
). In that same study we also showed that one of the ligands that Dan inhibits, Gdf5, is expressed by the Cajal-Retzius cells, but we were unable at that time to identify any functional significance for this pattern of expression (Kim and Pleasure, 2003
). Interestingly, the Cajal-Retzius cells are the most superficial cortical neurons, in Layer 1, and lie immediately adjacent to the cingulate pioneer neurons in the medial cortex.
To test the role of these factors, we examined the expression of Gdf5 and Dan in the mutant mice and found that Gdf5 is expressed in Cajal-Retzius cells in both control and mutant mice () and the expanded meninges in the mutant express abundant Dan (), implying that the levels of this inhibitor were increased in the vicinity. We also performed double labeling in the dorsal neocortex using Calretinin as a Cajal-Retzius cell marker (in the cingulate Calretinin stains both pathfinding neurons and Cajal-Retzius cells, but in the rest of the cortex Calretinin is a selective marker for Cajal-Retzius cells) and confirmed that Gdf5 was coexpressed by Cajal-Retzius cells, while Dan was expressed in the overlying meninges (). We then examined whether electroporation of Gdf5 in the midline of the cortex at E12.5 is sufficient to induce early Wnt3 expression by E14.5 and found that indeed Gdf5 electroporation induces low levels of Wnt3 expression in the medial cortex (). Thus, our model is that Wnt3 expression in the cingulate pioneer neurons is normally positively controlled by GDF5 activity from the adjacent Cajal-Retzius neurons but that the excess meningeally produced Dan in the mutant leads to decreased expression of Wnt3 in the mutant embryos. If our observations and model are correct, then overexpression of Dan in the cortical midline should also inhibit corpus callosum formation by antagonizing the actions of Gdf5. Indeed, expression of Dan led to delay of Calretinin+ pathfinding axons to cross the midline and failure of corpus callosum formation compared to control at E16.5 (), although this effect was apparently transient, because by E17.5, the callosum was formed in these mice (data not shown). This result implies that Wnt3 expression is finely controlled by neighboring cell types, which control the timing of corpus callosum formation by inducing the expression of Wnt3, allowing these axons to overcome the inhibitory effects of BMP7 from the meninges.
Figure 8 Control of Wnt3 expression by the interaction of Cajal-Retzius cells and meningeal cells. A – B′) In situ hybridization of Gdf5 (A) and Dan (B, B′) expression in the WT and Msx2-Cre; Ctnnb1lox(ex3) E15.5 heads. Gene expression (more ...)