Ectopic Expression of COUP-TFI in the Neocortex: Generation of D6/COUP-TFI Transgenic Mice
A 5.8-Kb genomic fragment upstream of mDach1
gene directs specific expression in the developing telencephalon beginning around E10.0 (van den Bout et al. 2002
). We took advantage of this genomic element to generate a transgenic mouse line that overexpresses COUP-TFI
in more dorsal parts of the cortical primordium (). Analysis of 2 independent lines (lines A and B) showed the same phenotype of cortical hypoplasia, and COUP-TFI
overexpression. Moreover, we found that the changes in expression level of the markers that we studied were reproducible in different litters over many generations (Supplementary Table 1
). Therefore, we focused our analysis on line A (haploid for the transgene). We found that the phenotype was extremely stable over many generations.
We began by defining the change in COUP-TFI
expression at E11.5, E12.5, E13.5, E15.5, E16.5, and E18.5 ( and data not shown). Ordinarily, at E11.5-E13.5, COUP-TFI
is expressed in cortical ventricular zone (VZ) progenitors in a ventralhigh
gradient (Qiu et al. 1994
; Liu et al. 2000
). This bidimensional gradient was disrupted in the D6/COUP-TFI cortex due to increased expression in the VZ of the dorsal pallium. We quantified the increase in expression by measuring the signal intensity using NIH Image J (see Materials and Methods). This analysis demonstrated a ~4-fold increase in COUP-TFI
expression in the VZ at E12.5 (zone 6, ). At E15.5, increased COUP-TFI
expression was also easily detectable in the CP (, arrows). We used this COUP-TFI
overexpression mutant, in combination with a COUP-TFI
null mutant, to dissect the role of this transcription factor in cortical development.
COUP-TFI Dosage Regulates Molecular Patterning of Cortical Progenitors
We examined the effect of modulating COUP-TFI dosage on the expression of transcription factors along the D/V dimension of the neocortical VZ (in the region of the somatosensory cortex primordium). We 1st focused on Pax6 and Emx2, both of which are known to regulate D/V patterning of the cortical VZ; Pax6 has strong effects ventrally (Yun et al. 2001
; Muzio et al. 2002
; Kroll and O'Leary 2005
), whereas Emx2 has its strongest effect in dorsal regions (Muzio et al. 2002
; Shinozaki et al. 2004
We found that overexpression of COUP-TFI began to reduce Pax6
expression by E11.5 (Fig. S1B
′); at E12.5, Pax6 expression was nearly extinguished (′). On the other hand, COUP-TFI loss of function led to increased Pax6 expression at E12.5, mainly in ventral regions (′). Emx2
expression was also affected by the changes in COUP-TFI dosage. Although Emx2
expression in D6/COUP-TFI cortex was not clearly affected at E11.5 (Fig. S1C
′), by E12.5 it was downregulated; on the other hand, it was upregulated in the COUP-TFI–/–
mutant (′). These results provide evidence that COUP-TFI is upstream of both Pax6
and that COUP-TFI regulates D/V patterning of the cortical progenitor zone. Both Pax6 and Emx2 are also implicated in regulating proliferation and differentiation (Warren et al. 1999
; Galli et al. 2002
; Estivill-Torrus et al. 2002
; Bishop et al. 2003
; Muzio et al. 2005
; Quinn et al. 2007
); thus we examined these parameters in the COUP-TFI mutants in the following sections.
Figure 2. COUP-TFI dosage regulates molecular patterning of cortical progenitors at E12.5. (A–A′) RNA in situ hybridization for COUP-TFI on coronal sections through the telencephalon of WT (A) and D6/COUP-TFI (D6) (A′), showing the change (more ...)
Tbr2 encodes a T-box transcription factor whose expression in the proliferative zone follows the normal ventrodorsal gradient in cortical neurogenesis (Bulfone et al. 1999
) and is thought to represent an intermediate stage in neural differentiation as cells mature to become Tbr1+
(Englund et al. 2005
). We compared Tbr2
expression in WT and D6/COUP-TFI brains from E11.5 to E13.5; during this interval, its expression in the mutant progenitor domains progressively increases (data not shown). At E12.5 in the D6/COUP-TFI cortex, Tbr2
was overexpressed in the dorsal proliferative zone (′). An opposite phenotype was found in the COUP-TFI–/–
mutant, where Tbr2
expression was reduced in ventral regions (′ insets).
Increased COUP-TFI Dosage Negatively Regulates Proliferation of Cortical VZ and Subventricular Zone Progenitors
We assessed the effect of COUP-TFI dosage on proliferation using different assays. The proliferation of progenitor cells is regulated largely by the progression of cells through the restriction point in late G1, and this critical step is regulated by the D-type cyclins such as Cyclin D2 (Sherr et al. 1994
). Cyclin D2
was strongly downregulated in the D6/COUP-TFI cortex, whereas it was upregulated in ventral regions of COUP-TFI–/–
cortex (″, arrowheads).
Figure 3. Increased COUP-TFI dosage negatively regulates proliferation of cortical VZ and SVZ progenitors. (A–A′′) RNA in situ hybridizations on coronal sections through the telencephalon of D6/COUP-TFI, COUP-TFI–/–, and (more ...)
To assess the VZ density of cells in M-phase of the cell cycle, we examined immunofluorescent labeling of phosphohistone-3 (PH3) at E11.5, E12.5, E13.5, and E15.5 (, , and S2). After quantification, D6/COUP-TFI cortex showed an ~2-fold reduced density of PH3+ VZ cells at E13.5 (′ and chart in C) and loss of COUP-TFI expression showed increased VZ proliferation by ~25% in ventral cortical regions (where COUP-TFI expression is the highest) at E13.5 (″ and chart in C). Thus, increases and decreases in COUP-TFI dosage were associated with reciprocal changes in Cyclin D2 expression and mitotic index.
Figure 4. Increasing COUP-TFI expression promotes neurogenesis. (A–F′) D6/COUP-TFI cortex shows increasing PP/CP thickness from E11.5 through E13.5 based on expression of β-III-tubulin (A–C′, red) and Tbr1 (D–F′). (more ...)
Next we examined the effect of changing COUP-TFI dosage on the secondary proliferative population, or basal progenitors, in the cortical subventricular zone (SVZ). SVZ progenitors are produced by the primary progenitors, radial glial cells (Kriegstein et al. 2006
). At E13.5, SVZ progenitors are detectable by their expression of PH3. In D6/COUP-TFI animals, there was a 2-fold reduction in PH3+
in all pallial regions where COUP-TFI is overexpressed (′ and quantification in D
), whereas in the COUP-TFI–/–
cortex there was an ~30% increase in ventral regions of the pallium (″ and quantification in D
), where normally COUP-TFI expression is strongest. By E15.5 both of these phenotypes were more severe (Fig. S2); almost no SVZ cells could be detected in the D6/COUP-TFI cortex (Fig. S2 A,A
To test whether it was possible to rescue the hypoproliferation phenotype in the D6/COUP-TFI cortex, we lowered COUP-TFI dosage by introducing the COUP-TFI null allele into these mice. D6/COUP-TFI;COUP-TFI–/– embryos showed a partial restoration of proliferation in both the VZ and SVZ (: compare ′ with ′″, arrowheads). In addition, there was a partial rescue of Pax6 expression (Fig. S3A–A′″, compare 3A′ with 3A′″). These results 1) demonstrate that COUP-TFI negatively regulates cortical proliferation and 2) confirm the specificity of the transgenic line. Thus increasing COUP-TFI expression promotes cell cycle exit, thereby depleting the pool of VZ/SVZ progenitors.
Increased COUP-TFI Dosage Positively Regulates Cortical Neurogenesis
To assess whether the COUP-TFI–induced changes in proliferation were coupled with changes in neurogenesis, we studied the expression of genes that are associated with the generation of cortical neurons.
Consistent with the increase in Tbr2 expression seen in D6/COUP-TFI mice, the cortical preplate (PP) and CP were thicker than in controls at E11.5, E12.5, and E13.5 measured by expression of β-III-tubulin (TUJ1) (′) and Tbr1 (′). The COUP-TFI–/– mutant showed a reciprocal phenotype—the thickness of Tbr1 expression in the ventral cortex was reduced (Fig. S3B and B″); the D6/COUP-TFI;COUP-TFI–/– mutants showed an intermediate phenotype (Fig. S3B–B′″; compare S3B′ with S3B′″). Moreover, we measured the PP/CP formation in D6 (, measurements performed in boxes in 3A′–C ′) and COUP-TFI–/– (, measurements performed in boxes in S3), and we found an initial outgrowth in D6 animals (, red line), followed by a reduced CP thickness after E13.5, showing a decrease of neuron-generating progenitors. On the other end, in COUP-TFI–/–, CP was always thinner than in the WT ().
Therefore, increasing COUP-TFI dosage appears to promote the switch between proliferation and neurogenesis at early stages of cortical development. This suggests that COUP-TFI can regulate the numbers of neurons generated at different stages of corticogenesis. Thus, we tested the laminar fate of cells leaving the cell cycle at different stages using the BrdU birthdating method. We administered BrdU to pregnant mice at E11.5, E13.5, and E16.5 and then analyzed the distribution of BrdU+ cells at P0 in WT and D6/COUP-TFI siblings (). At E11.5, in the D6/COUP-TFI mutants, there was an ~40% increase in the numbers of cells born (′ and quantification in , left); as in the WT, these migrated to the deep layers of the cortex. We did not observe a statistically significant change at E13.5 (′ and quantification in , middle). On the other hand, at E16.5, there was an ~70% reduction in the number of cells generated in the D6/COUP-TFI mutants (′ and quantification in , right); as in the WT, these migrated to superficial layers of the cortex. Thus, increasing COUP-TFI dosage shifts the ratio of deep to superficial neurons. This shift in laminar fate is coupled to a corresponding reduction in expression of Scip, a marker of layer 2/3 projection neurons, at P0 (Fig. S9A,A′), whereas markers of deep layer neurons persist (″). We did not find any changes in Reelin expression, a marker for Cajal-Reztius cells, during CP development (Fig. S9B,B′).
Figure 5. BrdU birthdating shows that increased COUP-TFI promotes generation of early-born cells and reduces late-born cells. (A–C′) BrdU injection at indicated stages, analysis at P0. BrdU immunostaining on coronal sections of WT (A–C) (more ...)
Figure 8. D/V patterning and layer V neurons are modified by COUP-TFI dosage. (A–A′) β-Galactosidase staining on coronal sections through the telencephalon of WT;BAT-gal (A), D6/COUP-TFI;BAT-gal (A′), and COUP-TFI–/– (more ...)
COUP-TFI Regulates the Fraction of Cells Leaving Cell Cycle
Our results suggest that increasing COUP-TFI dosage promotes neurogenesis of early-born neurons and the depletion of progenitors for late-born neurons. To determine whether altered COUP-TFI dosage is accompanied by altered probability of leaving the cell cycle, we measured the proportion of daughter cells that exit the proliferating population. We injected BrdU 12 h before analysis and performed double labeling for BrdU and Ki67, a marker of proliferative cells. We identified cells that had been proliferating, but had exited the cell cycle, as BrdU+;Ki67– (green); cells that had been in S-phase and remained in the cell cycle as BrdU+;Ki67+ (yellow); and cycling cells that had not been in S-phase as BrdU–;Ki67+ (red). We injected D6/COUP-TFI mice with BrdU at E11.5 (the 1st age at which we detected a reduction in proliferation). In the D6/COUP-TFI cortex, there was a clear increase in BrdU+;Ki67– cells, many of which were adjacent to the ventricle in the VZ, suggesting that they had just left the cell cycle (′, arrowheads). In the D6/COUP-TFI dorsal cortex, the proportion of cells that had left the cell cycle increased ~5-fold (). This change was not influenced by cell death, as we did not observe a change in apoptosis using activated Caspase-3 and TUNEL assay (Fig. S4).
Figure 6. COUP-TFI regulates the fraction of cells leaving the cell cycle. (A–A′) RNA in situ hybridization on coronal sections through the telencephalon at E11.5 showing COUP-TFI overexpression in the D6/COUP-TF cortex (box in A′): an equivalent (more ...)
We examined cell cycle exit in the COUP-TFI–/– mutant at E15.5 (the age where we identified increased proliferation; Fig. S2). In WT brains, cells that left the cell cycle (BrdU+/Ki67–) were visible at intermediate zone/SVZ transition (, arrowheads); the number of these cells was reduced in the COUP-TFI–/– mutant (′ and quantification in ). Together, the results show that COUP-TFI regulates the balance between proliferation and differentiation by controlling the probability that a dividing cell will exit the cell cycle and differentiate.
COUP-TFI Regulates RTK Signaling Pathways
Cortical regionalization, growth, and differentiation are regulated by secreted signals produced by patterning centers: Fgf rostroventrally and Bmp and Wnt dorsocaudally (reviewed in (Sur and Rubenstein 2005
). These signals control the expression gradients of several transcription factors, including COUP-TFI
(Fukuchi-Shimogori and Grove 2001
; Theil et al. 2002
; Garel et al. 2003
; Storm et al. 2006
). To determine how COUP-TFI promotes cell cycle exit, we investigated several signal transduction pathways implicated in this process in D6/COUP-TFI and COUP-TFI–/–
cortex. To obtain information about the primary phenotype, we started our analysis on the E11.5 cortex, soon after D6/COUP-TFI expression begins; at this time point, repression of Pax6
is evident (Fig. S1B
′). We examined the following signaling pathways that are associated with the proliferation/neurogenesis switch: Notch, Tgf-β, Bmp, Mapk/Erk, PI3-kinase/Akt, retinoid, and Wnt/β-catenin.
First, we assessed Notch signaling by examining expression of Hes5
, a gene induced by this pathway. COUP-TFI positively regulates Notch signaling (Tang et al. 2006
) and therefore could increase Hes5
expression. However, our analysis of Hes5
expression at E11.5 in the D6/COUP-TFI cortex showed no change in expression (Fig. S1E
′). At E13.5, Hes5
(Notch ligand) expressions in the cortical VZ were maintained (Fig. S5A
′), although the thickness of the VZ was reduced, consistent with the depletion of cortical progenitors that we previously observed. We did observe a reduction of Delta1
in the CP (Fig. S5A
′); however, we interpret this to be secondary to D/V patterning defects (see below).
In the developing cerebral cortex, Foxg1 and Bmp4 show reciprocal repression (Monuki et al. 2001
; Ohkubo et al. 2002
). We examined expression of Foxg1
, as this transcription factor promotes the proliferative state (Hanashima et al. 2002
; Martynoga et al. 2005
). At E11.5, we did not detect any change in Foxg1
expression in the D6/COUP-TFI cortex (Fig. S1D
′), suggesting that Bmp signaling may also not be affected. However, because Bmp’s and other Tgf-β proteins (i.e., activins) are implicated in promoting the switch from proliferation to differentiation (Ko et al. 1998
; Seoane et al. 2004
; Siegenthaler and Miller 2005
), we examined activation of Bmp and Activin signaling by assaying expression of phosphorylated Smad 1-5-8 and Smad 2, respectively (pSmad). As predicted by the normal Foxg1
expression, we did not observe a change in pSmad 1-5-8 and pSmad 2 at E11.5 in the D6/COUP-TFI cortex (Fig. S5G
RTKs, such as the Fgf receptors, are important regulators of many processes including patterning, differentiation, and proliferation (for review, see (Mason 2007
; Thisse and Thisse 2005
). Furthermore, Fgf8 signaling represses COUP-TFI
expression (Garel et al. 2003
; Storm et al. 2006
); thus we investigated whether COUP-TFI
gene dosage modification affected Fgf transduction pathways as well. We interrogated 2 Fgf-regulated pathways: Mapk/Erk kinase and PI3 kinase/Akt.
First, we examined activation of the Mapk/Erk kinase pathway. This kinase cascade culminates in phosphorylation and nuclear translocation of Erk1/2 and phosphorylates target transcription factors. We performed immunohistochemistry experiments using an antibody that detects phosphorylated forms of Erk1/2 (Corson et al. 2003
). At E11.5, pErk+
cells are detectable primarily in the cortical VZ: we found a clear reduction (50%) in the number of pErk+
cells in the D6/COUP-TFI mutants (′, boxes showing magnification on the right, and quantification in A
Figure 7. Regulation of RTKs and β-catenin signaling. (A–B′) Immunohistochemistry on coronal sections at E11.5 for pErk (A–A′) and Akt-pS (B–B′), showing the early response of RTK signaling to COUP-TFI overexpression. (more ...)
We next assayed the PI3 kinase/Akt pathway using an antibody that recognizes the substrates that are phosphorylated by Akt (Akt-pS) (Alessi et al. 1996
). At E11.5, we detected Akt-pS in cells lining the ventricle (′) and more diffusely throughout the VZ. In the D6/COUP-TFI mutants, Akt-pS immunoreactivity was diminished (30%) throughout the VZ (′, boxes showing magnification on the right, and quantification in B
At E13.5, assays for pErk and Akt-pS in the D6/COUP-TFI cortex confirmed the findings at E11.5. Furthermore, COUP-TFI–/– mutants showed complementary results for pErk and Akt-pS, providing additional evidence that COUP-TFI dosage indeed regulates these signaling pathways (′″). pErk staining prominently labeled VZ cells lining the WT ventricle (). In D6/COUP-TFI, these pErk+ cells were almost absent (′, quantification in ′″), whereas in COUP-TFI–/– there was a 25% increase (″, quantification in ′″).
In addition to labeling of VZ cells lining the WT ventricle, Akt-pS also labeled differentiating neurons in the CP (, asterisk). In the D6/COUP-TFI cortex, Akt-pS staining in the VZ was severely reduced (50%, quantification in ′″), whereas Akt-pS reactivity in the CP was greatly increased (′, asterisk). In COUP-TFI–/–, Akt-pS staining in the VZ and CP was slightly reduced (″ and quantification in ′″). These results suggest that loss of COUP-TFI expression increases Mapk/Erk signaling and decreases PI3K/Akt signaling. Furthermore, increasing COUP-TFI dosage suppresses pErk in the VZ and increases Akt-pS in the CP, further implying that these 2 signaling pathways are differentially regulated as COUP-TFI mediates the switch from proliferation to neural differentiation.
A link between COUP-TFI dosage and the Mapk/Erk–PI3K/Akt responses comes from analysis of Fgfr3
RNA expression; increased COUP-TFI increases Fgfr3
at E11.5 and E13.5 (′ and ′), whereas its expression is greatly reduced in the E13.5 COUP-TFI–/–
cortex (″). These results link decreased proliferation/increased neurogenesis with Mapk/Erk–PI3K/Akt inhibition and Fgfr3 activation. Studies on Fgfr3–/–
mice have associated signaling through Fgfr3
with reduced proliferation and increased differentiation of chondrocytes (Deng et al. 1996
; Sahni et al. 1999
) and pancreatic cells (Arnaud-Dabernat et al. 2007
), consistent with the phenotypes in the 2 COUP-TFI mutants. We did not find a change in Fgfr1
RNA expression in the D6/COUP-TFI cortex at E11.5 and E13.5 (Fig. S6A
We assessed Wnt signaling through the β-catenin pathway because of its known role in promoting proliferation and delaying neurogenesis (Chenn and Walsh 2002
; Soshnikova et al. 2003
; Backman et al. 2005
). We used the BAT-gal reporter transgene that reports canonical Wnt signaling tone (Maretto et al. 2003
). At E11.5, transgene activation is prominent in dorsocaudal parts of the cortical progenitor domain, the region that is known to have high levels of Wnt expression (Maretto et al. 2003
). In the D6/COUP-TFI mutant, β-catenin signaling was strongly decreased (′). At this stage, expression of the Frizzled8
) Wnt receptor is slightly decreased in the region of elevated COUP-TFI expression, perhaps contributing to the reduction in β-catenin signaling (Fig. S6C
′). Because Pax6
expression is nearly extinguished by increased COUP-TFI (′), we tested whether the reduction in Pax6
causes the reduced β-catenin signaling. We assayed the BAT-gal reporter in Pax6Sey/Sey
mice and found elevated Wnt signaling at E13.5 (Fig. S6I
′), contrary to the D6/COUP-TFI cortex. This demonstrated that COUP-TFI represses β-catenin signaling independent of its repression of Pax6.
Patterning Defects of the Dorsal Telencephalon
Taking into account the COUP-TFI expression gradient (ventralhigh
) and considering the disruption of this gradient in the D6/COUP-TFI and COUP-TFI–/–
mice, we tested the hypothesis that COUP-TFI also regulates cortical D/V patterning. First, we examined Wnt signaling in the CP with the BAT-gal transgene (Maretto et al. 2003
). At E15.5, this reporter shows a dorsalhigh
gradient of expression in the CP (). In the D6/COUP-TFI CP, BAT-gal expression is greatly reduced (′), whereas in the COUP-TFI–/–
mutant the expression expands ventrally (″). Thus, COUP-TFI regulates the D/V spatial distribution of Wnt/β-catenin signaling in the CP.
Figure 9. Model for COUP-TFI function in regulating proliferation, differentiation, and patterning. (A) COUP-TFI dosage regulates RTK signaling pathways through downregulation of Mapk/Erk and PI3K/Akt activity. These changes promote progenitor cells to leave the (more ...)
Next, we studied the distribution of gene expression markers of deep layers of the E15.5 CP. ER81 is a layer V marker that is expressed with a ventralhigh
gradient in the CP (). In D6/COUP-TFI cortex, ER81
was expanded into a more dorsal position, coincident with D6 overexpression (′). By contrast, in COUP-TFI–/–
expression shifted ventrally (″). This supports the idea that the high expression of COUP-TFI in the ventral cortical progenitor domain promotes ventral cortical identity. To test this further, we assessed the effect of modifying COUP-TFI dosage on the distribution of dorsal CP markers: Fezl
(layer 5) (Inoue et al. 2004
; Chen, Schaevitz, et al. 2005
; Chen, Rasin, et al. 2005
; Molyneaux et al. 2005
) and p75
(layer 6 and subplate) (Allendoerfer et al. 1990
). Indeed, increased COUP-TFI expression dorsally suppressed Fezl
expression, whereas reducing COUP-TFI
dosage led to ventral expansion of these markers (″). These results provide further evidence that COUP-TFI regulates cortical /V patterning.
COUP-TFI Regulates the Relative Numbers of Layer V Projection Neuron Subtypes
Layer V projection neurons have been classified according to their projection patterns: subcortically projecting (type I) and callosally projecting (type II) (Molnar and Cheung 2006
). These subtypes are distinguishable by their expression of different molecular markers, including ER81 and Fezl. Fezl is expressed in type I neurons, whereas ER81 is expressed in both types (Molnar and Cheung 2006
At E15.5, ER81+ cells appear in a ventrodorsal gradient, whereas Fezl+ cells are produced in a D/V gradient, suggesting that the type I and type II neurons are produced by distinct mechanisms. Indeed, COUP-TFI dosage differentially regulates their distributions (″). We examined the ER81 and Fezl expression at E18.5 to determine whether COUP-TFI dosage affected the final relative numbers of these deep cortical layer cells. We found that when COUP-TFI was overexpressed, ER81 was upregulated (′) and Fezl down-regulated (′). In contrast, loss of COUP-TFI function resulted in ER81 downregulation and Fezl upregulation (″). These results suggest that COUP-TFI can control the relative numbers of type I and type II projection neurons.