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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Neurosci. Author manuscript; available in PMC Aug 1, 2011.
Published in final edited form as:
PMCID: PMC3040956
NIHMSID: NIHMS270178

Wnt signaling regulates neuronal differentiation of cortical intermediate progenitors

Abstract

Cortical intermediate progenitors (IPs) comprise a secondary neuronal progenitor pool that arises from radial glia (RG). IPs are essential for generating the correct number of cortical neurons, however, the factors that regulate the expansion and differentiation of IPs in the embryonic cortex are largely unknown. In this study, we show that the Wnt-β-catenin pathway (canonical Wnt pathway) regulates IP differentiation into neurons. Upregulation of Wnt-β-catenin signaling by overexpression of Wnt3a in the neocortex induced early differentiation of IPs into neurons and the accumulation of these newly born neurons at the SVZ/IZ border. Long-term overexpression of Wnt3a led to cortical dysplasia associated with the formation of large neuronal heterotopias. Conversely, downregulation of Wnt-β-catenin signaling with Dkk1 during mid and late stages of neurogenesis inhibited neuronal production. Consistent with previous reports, we show that Wnt-β-catenin signaling also promotes RG self-renewal. Thus, our findings show differential effects of the Wnt-β-catenin pathway on distinct groups of cortical neuronal progenitors: RG self-renewal and IP differentiation. Moreover, our findings suggest that dysregulation of Wnt signaling can lead to developmental defects similar to human cortical malformation disorders.

Introduction

Projection neurons of the neocortex are generated from two progenitor pools, radial glia (RG) and RG-derived intermediate progenitors (IPs) (Molyneaux et al., 2007). Analyses of the behaviors of RG and IPs through the course of neurogenesis suggest that their patterns of behavior are coordinated. During early neurogenesis, the RG pool produces neurons via asymmetric divisions that replace the radial glial cell and produce one neuron. As neurogenesis proceeds, the RG instead produce an IP along with one replacement RG. IPs dramatically amplify the neurogenic pool prior to terminal neurogenic divisions (Noctor et al., 2007, 2008). Thus, as neurogenesis continues the RG pool is largely static while the IP pool expands so that IPs take over the bulk of neuron production.

The Wnt-β-catenin pathway is believed to be a key regulator of this developmental progression. Wnts regulate many developmental processes including cell proliferation, specification, differentiation and migration (Logan and Nusse, 2004; Clevers, 2006). In the canonical signaling pathway, Wnt ligands bind to Frizzled-LRP5/6 receptor complexes at the plasma membrane promoting gene expression through β-catenin and LEF/Tcf transcription factor activity (Logan and Nusse, 2004; Clevers, 2006). Loss-of-function studies in LRP6 and β-catenin decrease cortical neuronal production (Woodhead et al., 2006; Zhou et al., 2006). This phenotype is supported by gain-of-function studies that suggest that the pathway promotes self-renewal of neuroepithelial progenitors during early neurogenesis (Chenn and Walsh, 2002; Machon et al., 2007; Wrobel et al., 2007). For instance, expression of a dominant active form of β-catenin promotes RG self-renewal and inhibition of IP production (Wrobel et al., 2007). However, studies conducted during mid neurogenesis have yielded inconsistent results. Focal ablation of β-catenin promotes RG cell cycle exit and neuronal differentiation (Woodhead et al., 2006). This is consistent with Wnt signaling maintaining the progenitor state in early neurogenesis. Other studies contradict this result, demonstrating a role for the Wnt-β-catenin pathway in promoting neuronal differentiation through N-myc and the neurogenic transcription factor Ngn1/2 (Hirabayashi et al., 2004; Israsena et al., 2004; Kuwahara et al., 2010). Together, these findings implicate multiple roles for the Wnt-β-catenin pathway but also reveal that further studies are necessary to reach a clearer understanding of the functions of the pathway in neurogenesis. It is our hypothesis that the Wnt-β-catenin pathway regulates both proliferation and differentiation in vivo in a context dependent manner, likely separated by either progenitor state or developmental time. Importantly, some previous experiments suggest that the Wnt-β-catenin pathway specifically regulates IPs (Viti et al., 2003; Hirabayashi et al., 2004; Kuwahara et al., 2010), but there has been little direct consideration of this question.

We set out to determine whether the Wnt-β-catenin pathway regulates RG and IPs independently. We utilized secreted factors of the pathway, Wnt3a ligand or Wnt inhibitor Dkk1, to avoid the caveats of transcription factor manipulation including activation of signaling in cells not normally responsive to Wnt ligands and the cell adhesion consequences of β-catenin. Our study uncovered a novel role for the Wnt-β-catenin pathway in promoting IP differentiation and the formation of neuronal heterotopias.

Materials & Methods

Animals

In utero electroporations were performed on CD1 wildtype (Charles River Laboratories) and BAT-gal transgenic heterozygous (Maretto et al., 2003) mice. RNA in situ hybridization and X-gal reaction were performed on tissue obtained from CD1 and BAT-gal mice, respectively. All animal protocols were approved by the UCSF Institutional Animal Care and Use Committee.

In utero electroporation, expression plasmids and BrdU labeling

Electroporations were performed as previously described in Li et al. (2008). Electroporations were conducted on E13.5 or E15.5 embryos, allowed to continue gestation in utero and harvested either at E14.5, E16.5, E18.5 or P2. DNA solution (100–300nl of 2–3µg/µl plasmid/10%Trypan Blue/10mTris-HCl pH8.0) was injected in the left lateral ventricles of cerebral cortices. DNA solution was allowed to diffuse throughout the ventricle prior to exposing ventricles to electric field at 33V for E13.5 and 37V for E15.5. The following plasmids were used: M38 TOP-dGFP (R. Moon, Addgene plasmid 17114); pCIG (GFP) (Megason and McMahon, 2002); pCIG2 mouse Wnt3a (Wnt3a), subcloned with pCIG2 (Hand et al., 2005) and mouse Wnt3a cDNA cloned from Ambion cDNA library; pCIG chicken Wnt3a (Wnt3a) (Galli et al., 2004); pCIGLEF1-VP16, subcloned with pCIG and LEF1-VP16 (LEF1 fused to the herpes simplex virus VP16 transactivation domain-contact Guangnan Li for info, ude.fscu@il.tnarg); pCIGmouseDkk1 and pCIGΔaa29-48humanβ-catenin (provided by L.W. Burrus, SFSU) (Δaa29-48humanβ-catenin (Tetsu and McCormick, 1999)); pCImRFP (provided by J.L.R Rubenstein, UCSF). Water-solubilized BrdU was injected into the peritoneal cavity to a final dose of 50µg BrdU/g bodyweight.

Immunohistochemistry and X-gal reaction

Mouse brains were collected, fixed, cryoprotected, and coronally cryosectioned at 20µm using standard methods. Sections were immunolabeled and stained with X-gal using standard methods. The following primary antibodies were used: chicken anti-GFP (1/800, Aves Labs), mouse anti-BrdU (B44, 1/50, BD Biosciences), mouse anti-MAP2 (2a and 2b, 1/500, BD Pharmigen), mouse anti-NeuN (1/200, Chemicon), rabbit anti-Active Caspase3 (1/500, BD Pharmigen), rabbit anti-CDP/Cux1 (M-222, 1/200, Santa Cruz Biotechnology), rabbit anti-Tbr2 (1/500, Chemicon/Millipore), rabbit anti-Pax6 (1/200, Covance), rat anti-Ctip2 (25B6, 1/300–1/800, Abcam), rat anti-Ki67 (TEC-3, 1/300, Dako), Primary antibodies were detected with goat secondary antibodies conjugated to Alexa fluorophores (Invitrogen). β-galactosidase enzymatic activity was detected with X-gal substrate (Invitrogen).

In situ hybridization

Mouse brain sections were prepared as described for immunohistochemistry. RNA in situ hybridization was performed as described in Li et al. (2008). Axin2 DIG-labeled RNA probe was generated from pYX-Asc-mouseAxin2 (Open Biosystems cat. EMM1002-7496341).

Image analysis, quantification and statistical analysis

Images were captured with QI imaging CCD camera and QCapture Pro software (Burnaby, British Columbia Canada). For each separate experiment, three or more embryos were used for qualitative analyses (n≥3) and five or more embryos were used for quantitative analyses (n≥5) except for dGFP+ Tbr2+ cell counts in which 3 embryos were used (n=3). Embryos from two or more litters were used for each separate experiment. For cell counts at E16.5, BrdU+ or Tbr2+ cells within a 100µm wide neocortical column were counted. For cell counts at P2, Cux1+ or Ctip2+ cells within a 300µm wide neocortical column were counted. Length and thickness were quantified with the ImageJ measuring tool and by taking the average of 3 different measurements of adjacent regions per sample. Normalized data (un-Ep normalized) were normalized by dividing measurements or counts of the electroporated neocortex by measurements or counts of the unelectroporated neocortex of the same rostral-caudal level of the same brain. Quantified results were expressed as the mean±SEM for n given samples. Two-tailed Student’s t test with unequal variance was used to analyze data except for Dkk1 electroporations in which one-tailed tests were used. A value of p<0.05 was considered statistically significant.

Results

Ectopic Wnt3a causes cortical dysplasia and neuronal heterotopias

We analyzed the effect of excess Wnt-β-catenin signaling by expressing Wnt3a in the neocortex at E13.5, a time-point when RG symmetric self-renewal has already slowed to bypass the early strong effects on this process, and analyzed the results at P2 when neurogenesis and migration have primarily subsided. We chose to ectopically express Wnt3a for several reasons: (1) Loss-of-function mutation of Wnt3a leads to dramatic disruption of neocortical development (Lee et al., 2000). (2) Wnt3a, Wnt2b and Wnt8b are thought to generate the medial-high to lateral-low gradient of Wnt-β-catenin activity that has been implicated in the regulation of patterning and neurogenesis in the dorsal cortex. (3) Wnt3a has been shown to activate the Wnt-β-catenin pathway in many biological contexts. (4) Other Wnts with specific expression in the neocortex (Wnt5b, Wnt7a and Wnt7b) have not been correlated with roles in neurogenesis. Moreover, Wnt7a and Wnt7b have instead been associated with dendritic complexity and endothelial/vasculature regulation (Hall et al., 2000; Rosso et al., 2005; Daneman et al., 2009). (5) Expression of a secreted ligand of the Wnt pathway incorporates regulation from the extracellular space to the nucleus and should elicit effects on cells normally responsive to Wnt ligands unlike expression of transcription factors of the Wnt pathway.

We utilized in utero electroporation of plasmids to ectopically express GFP, as a control, or Wnt3a, in the developing mouse neocortex. We first tested whether electroporated Wnt3a upregulated the Wnt-β-catenin pathway in BAT-gal transgenic and wildtype embryos. The BAT-gal transgene is a Wnt-β-catenin pathway reporter allele with β-galactosidase under the control of LEF/Tcf responsive elements (Maretto et al., 2003). We detected electroporated regions by immunolabeling for GFP and the expression of the BAT-gal transgene by X-gal colorimetric reaction with β-galactosidase in coronal sections of mouse brains. Wnt3a, but not GFP, strongly upregulated the expression of the BAT-gal transgene (Fig. 1A–D). Moreover, it is clear that Wnt3a elicits a non-cell autonomous effect. Increased β-galactosidase activity was seen beyond electroporated regions (solid lines-electroporated region in Fig. 1C,D; dashed lines-non-cell autonomous activity in Fig. 1D). To confirm that Wnt3a upregulates endogenous signaling as well, we analyzed Axin2 mRNA expression, a direct target of the Wnt-β-catenin pathway, by RNA in situ hybridization. Wnt3a electroporation at E13.5, but not GFP, increased and laterally extended Axin2 mRNA expression by E14.5 (Fig. S1A,B). Thus, ectopic expression of Wnt3a in the neocortex upregulates the Wnt-β-catenin pathway within 24 hours of electroporation.

Figure 1
Ectopic Wnt3a causes cortical dysplasia and neuronal heterotopias

To test the ultimate effects of Wnt3a overexpression, we analyzed electroporated brains at P2, 8 days post electroporation. The GFP expressing cells in control brains were distributed throughout the cortical plate whereas many GFP+ cells in Wnt3a-electroporated brains were clustered in a mass adjacent to the ventricle (cortical plate (cp) GFP, cp/white brackets; ectopic mass GFP, heterotopia (ht)/red brackets; Fig. 1E,I). To investigate the nature of these cell clusters, we analyzed the expression of the neuronal markers NeuN, Ctip2 and Cux1 by immunolabeling. NeuN is a pan neuronal marker while CTip2 and Cux1 labels specific layers of the cortical plate. Ctip2 is expressed moderately by Layer VI and strongly by Layer V early-born/deep layer neurons (Fig. 1F,H) while Cux1 is expressed strongly by layer IV-II late-born/upper layer neurons (Fig. 1G,H). Labeling with NeuN (Fig. S2B,D), Ctip2 and Cux1 (Fig. 1J–L) showed that Wnt3a-induced cell masses are predominantly made up of neuronal cells. We further analyzed the composition of these heterotopias with the MAP2 antibody,a dendritic marker and found that these large heterotopias are densely composed of disorganized dendrites (Fig. S2F,H).

To determine how long-term induction of Wnt signaling affects the organization of IPs remaining in the cortex by P2, we analyzed the expression of Tbr2 by immunolabeling. In control brains, there were few remaining Tbr2+ cells found in the SVZ (arrows, Fig. 1M–O). However, in Wnt3a-electroporated brains there were many more Tbr2+ cells in disorganized clumps or organized in rosettes in an expanded SVZ adjacent to the heterotopic neuronal mass (rosettes, arrowheads, Fig. 1P–R). Thus, long-term upregulation of the Wnt-β-catenin pathway expands the population and dramatically alters the organization of these cells. These factors are consistent with characteristics that would lead to production of excess neurons. Furthermore, disorganization and rosette reorganization of IPs could cause newly born neurons to migrate incorrectly, which could lead to the disorganized cortical plate and periventricular heterotopia seen at P2.

The formation of an extra-neocortical neuronal mass larger than the normal epithelium suggests that Wnt3a ectopic expression induces excess progenitor proliferation but does not ultimately hinder neuronal differentiation. These results confirm a function for the Wnt-β-catenin pathway in promoting progenitor proliferation as previously been described (Chenn and Walsh, 2002; Logan and Nusse, 2004; Woodhead et al., 2006; Zhou et al., 2006; Wrobel et al., 2007). Moreover, it potentially implicates dysregulation of Wnt-β-catenin signaling pathway in the occurrence of neuronal migration disorders, as previously suggested (Zhou et al., 2006; Freese et al., 2010).

Wnt3a promotes expansion of RG and differentiation of IPs

To begin to determine the mechanism of Wnt3a effects that lead to the dramatic changes seen at P2, we examined the distribution of neuronal progenitors 3 days post electroporation, at E16.5. We immunolabeled for Pax6 to label RG and Tbr2 to label IPs. Since the thickness and density of cells of Pax6+ ventricular zone (VZ) appeared greater after Wnt3a-electroporation compared to control (yellow brackets, Fig. 2A,C), we measured the thickness of the VZ and counted Pax6+ VZ cells and determined that both the thickness and cell number of the VZ are significantly greater in Wnt3a-electroporated brains than in controls (VZ thickness, Control: n=6, avg. 1.05 unEp-normalized VZ thickness, SEM+/−0.016; Wnt3a: n=6, avg. 1.28 unEp-normalized VZ thickness, SEM+/−0.035, P value 0.0005; Wnt3a/Control ratio = 1.22. Pax6+ VZ cells, Control: n=6, avg. 1.08 unEp-normalized cell number/100µm column, SEM+/−0.030; Wnt3a: n=6, avg. 1.28 unEp-normalized cell number/100µm column, SEM+/−0.067, P value 0.03; Wnt3a/Control ratio = 1.18; Fig. S3). This is consistent with Wnt3a inducing RG to undergo self-renewing divisions, an established function of the Wnt-β-catenin pathway.

Figure 2
Wnt3a promotes expansion of RG and differentiation of IPs

Next we analyzed the distribution of Tbr2+ cells. We demarcated the expression pattern of Tbr2 into two domains: the “deep Tbr2 domain” (DTD), consisting of Tbr2+ cells within the VZ and SVZ (Tbr2 domain below the arrowheads in Fig. 2B), and the “upper Tbr2 domain” (UTD) consisting of the more sparsely distributed Tbr2+ cells in the IZ (Tbr2 domain above the arrowheads in Fig. 2B). Strikingly, Wnt3a electroporation led to a dramatic depletion of UTD IPs compared to control (Fig. 2B,D). There are several potential cellular mechanisms for this change: Wnt3a may 1) block production of IPs from RG; 2) decrease proliferation of IPs; 3) block transition of IPs from the DTD to UTD; 4) induce early differentiation of IPs into neurons; 5) force IPs to become a non-neuronal Tbr2− cell population.

Normally, an increase in RG proliferation and population would be expected to lead to increased production of IPs. However, we observed a clear decrease of IPs in the UTD of experimental samples (Fig. 2B,D). It seems likely that induction of RG proliferation by Wnt3a might also be associated with inhibiting transition of RG into the IP state, thus reducing the total IP population. Previous work by Wrobel and colleagues (2007) has shown that genetically inducing RG proliferation with a dominant active allele of β-catenin (ΔEx3β-catenin) inhibits the production of IPs. To test whether this is the explanation for loss of IPs after Wnt3a electroporation, we quantified the number of Tbr2+ cells in the total and Tbr2 subdomains (DTD and UTD) (Fig. 2E,F; quantification: Control N=7, Wnt3a N=9, Fig. 2G). Since IPs should be generated and seen first in the DTD (at the VZ-SVZ border), the numbers of cells in the DTD should be reduced if there is a block in IP production from RG. Indeed, the total number of Tbr2+ cells is significantly decreased in Wnt3a-electroporated brains compared to controls (Total, Control: avg. 207 cells/100µm column, SEM+/−11.6; Wnt3a: avg. 172 cells/100µm column, SEM+/−9.7, P value 0.037; Fig. 2G), but the number of Tbr2+ cells in the DTD is unchanged (DTD, Control: avg. 139 cells/100µm column, SEM+/−5.6; Wnt3a: avg. 151 cells/100µm column, SEM+/−7.8, P value 0.219; Fig. 2G). Instead, there is a dramatic decrease in UTD IPs after Wnt3a electroporation compared to control (UTD, Control: avg. 69 cells/100µm column, SEM+/−7.6; Wnt3a: avg. 21 cells/100µm column, SEM+/−5.6, P value 0.0003; Fig. 2G). Thus, the decrease in total Tbr2+ cells is more likely due to the depletion of UTD Tbr2+ cells rather than a failure to generate DTD Tbr2+ cells from RG.

The loss of Tbr2+ cells in the UTD could be due to a failure of IPs to continue to proliferate in this zone. To assess this, we calculated the mitotic fraction of Tbr2+ cells in the DTD and UTD by acute BrdU labeling (50µg BrdU/g body weight 1hr before harvest at E16.5). We quantified the total, DTD and UTD fraction of Tbr2+ cells that are BrdU+ (Fig. 2E,F; quantification: Control N=7, Wnt3a N=6, Fig. 2H). The S-phase fraction of DTD Tbr2+ cells in Wnt3a-electroporated brains is similar to controls (DTD, Control: avg. 0.086/100µm column, SEM+/−0.0077; Wnt3a: avg. 0.084/100µm column, SEM+/−0.0062, P value 0.828; Fig. 2H). Interestingly, although the number of UTD Tbr2+ cells is dramatically decreased, the S-phase fraction of UTD and total Tbr2+ cells after Wnt3a electroporation is also similar to controls (UTD, Control: avg. 0.347/100µm column, SEM+/−0.052; Wnt3a, avg. 0.335/100µm column, SEM+/−0.094, P value 0.913; Total, Control: avg. 0.170/100µm column, SEM+/−0.019; Wnt3a, avg. 0.123/100µm column, SEM+/−0.015, P value 0.080; Fig. 2H). Thus, although there is a dramatic loss of UTD IPs, the level of proliferation of IPs is maintained.

To test if UTD IPs are absent because they have stopped expressing Tbr2, we traced the lineage of IPs by pulsing with BrdU 24hrs before harvesting embryos. This method labeled progenitors that are in the S-phase at E15.5 and allowed us to visualize their location at E16.5. If the absence of UTD IPs is not due to failed production of this population but is due to downregulation of Tbr2 expression, we would see an extra pool of BrdU+ cells that are Tbr2− in Wnt3a-electroporated regions but not in controls. Indeed, we observed such a large pool of BrdU+, Tbr2− cells in the apical-most region of the UTD of Wnt3a-electroporated brains but not in controls (Fig. 2I,M). To test if the BrdU+, Tbr2− pool of cells are no longer dividing or have just lost Tbr2 expression, we immunolabeled with Ki67, an active-cell cycle marker. This confirmed that the BrdU+, Tbr2− cells found in the UTD are also Ki67- and, thus, are no longer proliferative (Fig. 2J,K,N,O).

The disappearance of UTD IPs and the concomitant appearance of BrdU+, Tbr2− post mitotic cells in the UTD suggest that ectopic Wnt3a induces UTD IPs to become post mitotic. To confirm if the BrdU+, Tbr2− cell population are differentiated neurons, we immunolabeled with Ctip2 antibody. At E16.5, Ctip2 is only faintly expressed in some cells in progenitor domains but relatively more strongly expressed by post-migratory, differentiated neurons localized in the cortical plate. We found that electroporation of Wnt3a but not GFP lead to increased expression of Ctip2 in progenitor domains. Moreover, a large number of the UTD-accumulated BrdU+, Tbr2− cells also express Ctip2 (Fig. 2L,P). Thus, upregulation of the Wnt-β-catenin pathway with Wnt3a induces UTD IPs differentiate into neurons.

The IP pool is critical for generating the correct number of cortical plate neurons. Intermediate progenitors amplify the neuronal output of RG by providing 1 or 2 extra steps of self-renewing divisions prior to producing a pair of neurons on its last symmetric division. We therefore hypothesized that premature differentiation of IPs and accumulation of the resulting neurons in the UTD in Wnt3a-electroporated regions would lead to a reduction in the number of neurons reaching the cortical plate during this period. To address this hypothesis, we examined Ctip2 and Cux1 expression by immunolabeling at E16.5, 3 days post electroporation. Within the cortical plate, the expression patterns of both markers were reduced in Wnt3a-electroporated regions compared to controls (white dashed lines, Fig. 2Q–X). The population of late-born/upper layers neurons labeled by Cux1 was more strongly affected, being either absent or thinner in Wnt3a-eletroporated regions relative to the unaffected regions and controls (white dashed lines, Fig. 2S,T,W,X). To quantify these observations, we measured the thickness of both Ctip2 and Cux1 cortical plate layers and determined that these layers in Wnt3a-electroporated brains are significantly thinner than controls (Ctip2 layer thickness, Control: n=6, avg. 1.07 unEp-normalized thickness, SEM+/−0.031; Wnt3a: n=6, avg. 0.66 unEp-normalized thickness, SEM+/−0.050, P value 0.00009; Wnt3a/Control ratio = 0.61. Cux1 layer thickness, Control: avg. 0.96 unEp-normalized thickness, SEM+/−0.037; Wnt3a, avg. 0.39 unEp-normalized thickness, SEM+/−0.004, P value 0.00009; Wnt3a/Control ratio = 0.41; Fig. S4). To rule out a significant contribution of cell death to this effect, we immunolabeled for active Caspase3, a marker of cell death, and observed no significant increase of apoptosis in Wnt3a-electroporated brains compared to controls (Fig. S5A–D). These results suggest that premature differentiation of IPs and accumulation of these newly born cells in the UTD in Wnt3a-electroporated brains lead to reduction in the number of neurons reaching the cortical plate.

Newborn neocortical neurons use RG processes as a migratory scaffolding, therefore, the failure of newly-born neurons to reach the cortical plate might also be attributed to loss of RG cell integrity. We analyzed the structure of the radial processes using Nestin antibody. Nestin is localized in the cell bodies, extensions and endfeet of RG cells, thus allowing examination of RG extensions and pial connection of their endfeet. The general appearance of Nestin immunolabeling is similar between control and experimental (Fig. S6B,E). Moreover, the appearance of extensions in the cortical plate and endfeet at the pial surface look unperturbed by Wnt3a overexpression (Fig. S6C,F). Thus, our analyses indicate that perturbation of RG morphology and pial connection is likely not a primary factor in the formation of Wnt3a-induced heterotopias.

Lateral expansion of the neocortical epithelium is achieved by self-renewal of RG. At E16.5, the neocortices electroporated with Wnt3a are generally longer than controls (yellow dashed lines, Fig. 2Q,U). In addition, in regions electroporated with Wnt3a the cortical wall was frequently thinner than regions distant from the site of electroporation and in controls (white double-ended arrows, Fig. 2Q,U; solid line, Fig. 1A–D). We measured the length and thickness of neocortices and determined that Wnt3a-electroporated neocortices were significantly longer and thinner than controls (Neocortex length, Control: n=6, avg. 0.98 unEp-normalized length, SEM+/−0.036; Wnt3: n=6, avg. 1.38 unEp-normalized length, SEM+/−0.063, P value 0.0006; Fig. S7A; Wnt3a/Control ratio = 1.41. Neocortex thickness, Control: n=6, avg. 1.02 unEp-normalized thickness, SEM+/−0.016; Wnt3a: n=6, avg. 0.79 unEp-normalized thickness, SEM+/−0.060, P value 0.011; Fig. S7B; Wnt3a/Control ratio = 0.78). This morphology is consistent with the abnormal expansion of the RG pool observed at E16.5 (Fig. 2A,C) in Wnt3a-electroporated brains and may ultimately contribute to the production of excess neurons observed at P2 once this increased cohort of RG produces neurons.

Intermediate progenitors are targets of Wnt signaling in vivo

If the Wnt-β-catenin pathway is physiologically important for IP differentiation, then endogenous Wnt-β-catenin signaling must be active in IPs. To test whether IPs are responding to endogenous Wnts, we electroporated a TOP-destablizedEGFP (TOPdGFP) Wnt-β-catenin pathway reporter plasmid into E13.5 brains and analyzed expression of dGFP at E14.5. First, to test the relative expression of dGFP in expressing cells we co-electroporated TOPdGFP with a control plasmid ubiquitously expressing RFP, (pCImRFP). Since RFP is under the control of a constitutive promoter, the level of RFP expression serves as a visual measure of the relative electroporation efficiency. We observed cells strongly expressing RFP that were either weakly or strongly expressing dGFP (Fig 3A1,A4). Similarly, we observed cells weakly expressing RFP that were either weakly or strongly expressing dGFP (Fig 3A2,A3). This expression heterogeneity suggests that the Wnt reporter expression differences that we observe are not solely due to differences in plasmid number but rather probably readout of endogenous levels of Wnt signaling. Thus, as others have also concluded (Woodhead et al., 2006), the TOPdGFP plasmid introduced into the neocortex by electroporation can be used to identify the presence of endogenous Wnt-β-catenin signaling.

Figure 3
Intermediate progenitors can be targets of Wnt signaling in vivo

We next analyzed the colocalization of Tbr2 and dGFP to determine if IPs are included among the cells responsive to endogenous Wnt-β-catenin signaling. Many Tbr2 expressing cells were dGFP+ (arrows, Fig. 3B). In the VZ few Tbr2+ cells were also dGFP+ (Fig. 3B1), while in the most basal area of the DTD many more are doubly expressing Tbr2 and dGFP (Fig. 3B2). By E14.5 some Tbr2+ cells have emerged from the DTD and migrated into the UTD (Fig. 3B). These UTD Tbr2+ cells also express dGFP (Fig. 3B3). We quantified the number of dGFP+, Tbr2+ cells that are localized within these domains and verified that IPs within the basal DTD are most actively responding to endogenous Wnt-β-catenin signaling at E14.5 (dGFP+, Tbr2+ cells, n=3, VZ: avg. 14% cells/100µm column, SEM+/−5.56; Basal DTD: avg. 69% cells/100µm column, SEM+/−1.42; UTD: avg. 17% cells/100µm column, SEM+/−4.71; Fig. 3C). These observations indicate that Tbr2-expressing IPs respond to endogenous Wnt-β-catenin signaling.

Wnt3a promotes differentiation of neurons in progenitor domains and disorganization of RG and IP distribution

The BrdU+, Tbr2−, Ctip2+ post mitotic cells that accumulated in the UTD in Wnt3a-electroporated regions are likely immature neurons born from the missing UTD IP population. To determine if these cells will mature into the neurons that later comprise the heterotopia seen at P2, we looked for ectopic expression of neuronal markers at E18.5 (5 days post electroporation). Normally Ctip2 expression is only strong in layers VI and V of the cortical plate at E18.5 (Fig. 4B,D). However, in Wnt3a-electroporated regions there was ectopic expression of Ctip2 in progenitor domains (ht, Fig. 4B,D,F,H). Cux1 is typically expressed by some cells in progenitor domains but mostly by differentiated neurons in layer IV-II of the cortical plate at E18.5 (Fig. 4C,D). However, in Wnt3a-electroporated regions Cux1 expression was dramatically expanded in the progenitor/intermediate zone (IZ) domains and decreased in the cortical plate ((Fig. 4C,D,G,H). Together with our previous findings, our results suggest that ectopic Wnt3a induces premature differentiation of UTD IPs and accumulation of these cells in progenitor domains. This hypothesis is consistent with the decreased thickness of the cortical plate and increased thickness of the heterotopic region (cp/white double-ended arrows, ht/yellow double-ended arrows, Fig. 4A–H; cp/white brackets, ht/red brackets, Fig. 1E,I).

Figure 4
Wnt3a promotes differentiation of neurons in progenitor domains

To further understand the progression of heterotopia formation, we analyzed the distribution of RG and IPs at E18.5, 5 days post Wnt3a-electroporation. As before, we used Pax6 to label RG and Tbr2 to label IPs. Using these immunomarkers we noted scattering of progenitors and reorganization of progenitors into rosettes (scattered cells-arrows; rosettes-arrowheads, Pax6, Fig. 5A–D; Tbr2, Fig. 5I–L). We further tested if RG and IPs in Wnt3a-electroporated brains remain active in the cell cycle by co-immunolabeling with Ki67. We observed that a large proportion of RG and IPs are Ki67+ and, thus, are actively in the cell cycle (Pax6, Fig. 5E–H; Tbr2, Fig. 5M–P). Thus, Wnt3a overexpression leads to premature differentiation of IPs to neurons and disorganization of progenitor distribution without completely depleting the RG and IP pool.

Figure 5
Wnt3a promotes disorganization of RG and IP distribution

Dominant activation of Wnt signaling drives ectopic neuronal differentiation

To determine whether cell autonomous activation of Wnt signaling leads to similar effects as ectopic Wnt3a expression, we tested whether a strong dominant active form of LEF1 could elicit the same effects. The LEF/Tcf family of transcription factors transduce the transcriptional output of the Wnt-β-catenin pathway. We used a dominant active form of LEF1 fused to the transactivation domain of the VP16 protein from the herpes simplex virus (LEF1-VP16). Consistent with the effects of Wnt3a electroporation, LEF1-VP16 promoted the differentiation of Tbr2+ IPs in the UTD 3 days post-electroporation (Fig. 6A–P). The cohort of LEF1-VP16-electroporated cells did not express Tbr2 (arrowsheads, Fig. 6B–D,F–H) or Ki67 but strongly expressed Ctip2 (arrows, Fig. 6I–P). We saw similar effects after expression of a dominant active form of β-catenin (Δ29-48βcat) (arrows, Fig. S8A–H).

Figure 6
Dominant activation of Wnt signaling drives ectopic neuronal differentiation

Dkk1 inhibits the production of neurons during mid and late neurogenesis

To determine whether Wnt signaling is required for the normal production of neurons in the neocotex, we ectopically expressed Dkk1, a secreted antagonist of the Wnt-β-catenin pathway coreceptor LRP6, and analyzed the production of neocortical neurons. We used Ctip2 to label early-born/deep layer neurons and Cux1 to label late-born/upper layer neurons. We then counted the numbers of neurons of Layer V and Layer IV-II, neurons primarily born after the time-point of electroporation at E13.5. Ectopic expression of Dkk1 at E13.5 (Fig. 7A–H) inhibited the production of Layer V neurons but not the production of Layer IV-II neurons (Ctip2, Control: N=6 avg. 92 cells/300µm column, SEM+/−7.06; Dkk1: N=5, avg. 69 cells/300µm column, SEM+/−7.37, P value 0.027, Fig. 6I; Cux1, Control: N=6 avg. 792 cells/300µm column, SEM+/−23.45; Dkk1: N=5, avg. 744 cells/300µm column, SEM+/−48.55, P value 0.186; Fig. 7J). On one hand this result suggests that Dkk1 might selectively regulate deep layer neuron production. However, we wondered whether instead the effects of Dkk1 were merely transient but general so that if electroporated later it would inhibit superficial neuron generation instead. To test this, we expressed Dkk1 at E15.5 (Fig. 7K–R) and found that it specifically inhibited production of Layer IV-II (Ctip2, Control: N=6 avg. 108 cells/300µm column, SEM+/−5.20; Dkk1: N=6, avg. 107 cells/300µm column, SEM+/−3.80, P value 0.487, Fig. 7S; Cux1, Control: N=6 avg. 595 cells/300µm column, SEM+/−35.09; Dkk1: N=6, avg. 514 cells/300µm column, SEM+/−21.86, P value 0.043; Fig. 7T). These results suggest that inhibition of Wnt signaling by Dkk1 generally inhibits neurogenesis without specifically regulating different layer specificities and are consistent with the phenotype of the Wnt3a electroporation experiments: The agonist Wnt3a generates extra neurons while the antagonist Dkk1 inhibits the production of neurons. Moreover, since IPs generate the majority of neurons after E15.5, our results further imply a role for the Wnt-β-catenin pathway in the regulation of IP behavior.

Figure 7
Dkk1 inhibits the production of neurons during mid and late neurogenesis

Discussion

The IP stem cell pool is critical for the generation of the correct numbers and subtypes of neurons during mammalian neurogenesis. In this study we show that the Wnt-β-catenin pathway is active in neocortical IPs and required for production of projection neurons during mid and late stages of neurogenesis. We further show that the Wnt-β-catenin pathway plays a pivotal role in regulating IP differentiation and that dysregulation of the pathway can lead to cortical dysplasia and the formation of neuronal heterotopias.

The Wnt-β-catenin pathway regulates IP differentiation

Our data shows that induction of the Wnt-β-catenin pathway with excess ligand leads to early differentiation of IPs into neurons. These neurons fail to migrate to the cortical plate and accumulate within the UTD (Fig. 8A). Together these findings indicate that the Wnt-β-catenin pathway regulates the timing or progression of IP differentiation. These findings have not previously been described by other in vivo studies, perhaps because other studies relied solely on expression of intracellular signaling molecules that may have additional cell-autonomous consequences. Like our results, previous in vitro studies did show that Wnt-β-catenin signaling induces neuronal differentiation in neural precursor cell cultures (Hirabayashi et al., 2004). Moreover, the promoters of the proneuronal bHLH transcription factors, neurogenin1 (Ngn1) and neurogenin2 (Ngn2) contain LEF/Tcf elements and can be activated by β-catenin in vitro (Hirabayashi et al., 2004; Israsena et al., 2004), and loss-of-function mutation of β-catenin leads to loss of Ngn2 expression (Backman et al., 2005). In accord with these previous findings, our data further implicates the Wnt-β-catenin pathway in specifically regulating the timing or progression of differentiation of IPs into projection neurons.

Figure 8
The Wnt-β-catenin pathway regulates RG self-renewal and IP differentiation

Superficially there appears to be a contradiction between the ample evidence that the Wnt-β-catenin pathway promotes symmetric progenitor proliferation during early and mid neurogenesis (Chenn and Walsh, 2002, 2003; Zechner et al., 2003; Woodhead et al., 2006; Wrobel et al., 2007) and the studies showing that, beginning at mid neurogenesis, the Wnt-β-catenin pathway promotes differentiation of neuronal progenitors (Hirabayashi et al., 2004; Israsena et al., 2004; Hirabayashi and Gotoh, 2005; Guillemot, 2007). Retroviral infection of RG with either S33Yβcat or Wnt7a led to enrichment of infected cells in the SVZ (Viti et al., 2003; Kuwahara et al., 2010). These affected cells could be arrested in the SVZ due to early differentiation of IPs, similar to what we have observed with our results. Thus, our results along with other studies suggest that the Wnt-β-catenin pathway regulates both self-renewal and differentiation but in different pools of progenitors: self-renewal in RG and differentiation in IPs (Fig. 7B).

Complementing our findings using excess Wnt ligand, we found that downregulation of endogenous Wnt-β-catenin signaling with Dkk1 inhibits neuronal production during mid and late stages of neurogenesis. This implies a required role for the Wnt-β-catenin pathway in the regulation of IP behavior, since IPs generate a large proportion of neurons during mid neurogenesis and may function as the only neuron-generating progenitor pool during late neurogenesis (Noctor et al., 2007, 2008). Downregulation of endogenous Wnt-β-catenin signaling with Dkk1 likely reduces the number of IPs that differentiate over a given time period, leading to less neurons produced over the course of neurogenesis.

The differential effects of both Wnt3a and LEF1-VP16 on the UTD subpopulation of IPs suggest that the DTD and UTD IP subpopulations have some distinctions. A dramatic loss of UTD IPs that coincided with the formation of ectopic neurons in the UTD was observed in Wnt3a-electroporated brains, but the size and proliferation level of DTD IPs were unaffected. Thus, it appears that there is specificity in the response of UTD IPs to Wnt-β-catenin signaling. We defined the subpopulations of Tbr2+ IPs into the DTD and UTD domains in this study based on the Wnt3a phenotype we observed as there are currently no known molecular markers that define the DTD and UTD IP subpopulations. However, based on our results there must be some cellular distinctions between these subpopulations of IPs, although they may have a precursor-product relationship.

Excess Wnt signaling leads to cortical dysplasia and neuronal heterotopias

Long-term upregulation of the Wnt-β-catenin pathway with Wnt3a resulted in cortical dysplasia associated with the formation of large neuronal heterotopias. The heterotopias are composed of both early-born/deep layer and late-born/upper layer projection neurons and contained disorganized axonal (data not shown) and dendritic processes (Fig. S2F,H). These features are similar to those found in cortical malformations of patients (Fox and Walsh, 1999; Lu and Sheen, 2005; Lian and Sheen, 2006). Thus, our findings indicate possible roles for the Wnt-β-catenin pathway in cortical malformation disorders.

We observed several factors that likely contributed to these findings in the Wnt3a-electroporated brains. Ectopic differentiation of IPs in the UTD is most likely the direct factor for the formation of ectopic neurons in progenitor domains. We observed the stepwise expansion of the pool of ectopic neurons in progenitor domains from E16.5 to E18.5 to P2. Secondly, the abnormal horizontal expansion of RG, observed at E16.5, would eventually lead to extra IPs and neurons. To this end, a dramatically larger population of Tbr2+ IPs is present at P2 in Wnt3a-electroporated brains compared to controls. Third, we observed in some samples Ctip2+ nodules protruding at the ventricular surface of the neocortex of Wnt3a-electroporated brains at E16.5 (asterisks, Fig. 2U–X). These are likely RG that have directly differentiated into neurons within the VZ. Lastly, disorganization and reorganization of RG and IPs into rosettes, which we observed at E18.5 and P2, could lead to disruption of migration of daughter neurons to the cortical plate.

Other studies demonstrating that Wnt-β-catenin signaling induces differentiation of neocortex-derived neural precursor cell cultures (Hirabayashi et al., 2004) and regulate the expression of Ngn1/2 proneuronal genes (Hirabayashi et al., 2004; Israsena et al., 2004; Backman et al., 2005) support our conclusion that the primary consequence of Wnt-β-catenin pathway activation is the regulation of neocortical IPC differentiation . However, the dramatic effect we see on the position of neurons raises the possibility that there is a direct role for canonical Wnt signaling on cell motility, as shown in other contexts (Moon et al., 1993; Silhankova and Korswagen, 2007). Upregulation of Wnt-β-catenin signaling might directly inhibit IP or newborn neuron migration. It is also possible that the expression of Wnt3a also has effects on the non-canonical Wnt signaling pathway, which can also have direct migratory consequences. Future experiments that can differentiate between direct and indirect effects on migration or the neural differentiation program will be necessary to clarify this matter.

In conclusion, we’ve defined new roles for the Wnt signaling pathway in controlling the terminal differentiation of IPs and shown that dysregulation of this signaling axis can lead to cortical malformations similar to those seen in some patients. This will make it attractive in the future to consider the Wnt signaling pathway as a target for mutations in the inherited disorders of cortical migration.

Supplementary Material

Supp1

Acknowledgements

This work was supported by K02 MH074958, R01 MH066084, the National Multiple Sclerosis Society and a generous gift endowment from the family of Glenn W. Johnson, Jr.

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