Reduction of Cortical Neurons' Numbers Especially in Superficial Layers upon CP Transient Neurons Ablation
We have previously shown that the specific ablation of the CP transient neurons derived from Dbx1
-expressing progenitors at the VP/PSB in E1-Ngn2/CRE(iresGFP);Dbx1DTA
mutant animals leads to a ~20% reduction in CP thickness throughout the neocortex at E18.5, without affecting either cell density or death (Teissier et al. 2010
). This population invades the preplate (PP) and the SVZ/intermediate zone (IZ) by tangential migration with a caudolateralhigh
gradient starting at E12.5 and is redistributed homogeneously in the CP along the rostrocaudal (RC) and mediolateral (ML) axes at birth.
In order to understand how the loss of Dbx1
-derived CP transient neurons leads to a reduction of the CP thickness in E1-Ngn2/Cre(iresGFP);Dbx1DTA
animals, we analyzed the defects at caudolateral (cL) and rostrodorsal (rD) levels which represent cortical territories at short and long distances from their generation site, respectively (Teissier et al. 2010
). Nissl staining analysis at P2 confirmed that the entire cortex showed a decrease in CP thickness, as previously described at E18.5, with respect to wild-type cortices (rD: 24.32% and cL: 23.87%) (Teissier et al. 2010
and data not shown) and also revealed that this reduction was clearly more pronounced for superficial layers (, rD: 38.98%; cL: 35.77%) than for deep layers (rD: 17.38%; cL: 16.04%) at both rD and cL levels. No significant differences were observed in the extent of the defects between cL and rD levels. These results were confirmed by specifically labeling deep and superficial layers using Tbr1 and Cux1/2 immunostaining, respectively. We observed in both cL and rD regions a small decrease in the thickness of Tbr1+
deep layers in addition to a severe reduction in that of Cux1/2+
superficial layers in mutant brains (). Laminar fate and positioning of cortical neurons appeared to be preserved as observed by immunohistochemistry for FoxP2 (deep layers) and Ctip2 (prospective layer V subpopulations) () as well as in situ hybridization for Cdh8
(prospective layers II–IV and subpopulation of layer V) and Rorβ
(prospective layer IV) (). In addition, an increase in the number of FoxP2 neurons was detected in deeper portions of layer VI (, white bars), whereas that of neurons fated to layer V, as identified by Ctip2 labeling (), was slightly decreased in mutant compared with control littermates. A more pronounced decrement of Cdh8
staining confirmed that superficial layers are more highly affected as revealed by Cux1/2 labeling (). Finally, since another population of tangentially migrating cells, namely Cajal–Retzius (CR) cells, are also generated from Dbx1
-expressing progenitors at the VP prior to the CP transient neurons and have been shown to influence cortical patterning (Griveau et al. 2010
), we performed whole-mount in situ hybridization for Lmo4
mRNAs on P0 animals (Supplementary Fig. S1
). We observed no differences in area patterning between ablated and control littermates cortices as expected from the absence of defects in CR cells distribution previously reported in the E1-Ngn2/CRE;Dbx1DTA
mutants (Teissier et al. 2010
) and the late migration and homogeneous distribution of the CP transient neurons.
We conclude that the specific genetic ablation of the CP transient glutamatergic neurons generated from Dbx1-expressing progenitors in the VP leads to a homogeneous deficit in the total number of neurons, although this is more pronounced for the superficial layers, throughout the early postnatal neocortex.
Defects in Differentiation and Proliferation Progressively Affect the Entire Pallium along the Expected Trajectory of the Ablated CP Transient Neurons
Since the CP transient neurons progressively invade the pallium from caudolateral regions starting at E12.0 and reach the rostrodorsal territories by E14.5 (Teissier et al. 2010
), we began by analyzing neuronal differentiation at E12.5 and E14.5 using immunostaining for TuJ1 and Tbr1, markers of young postmitotic neurons (Memberg and Hall 1995
) and early differentiated glutamatergic cortical neurons (Englund et al. 2005
), respectively. We observed an increase in the number of both Tbr1+
differentiating neurons in the CP (, black bars) together with ectopically positioned labeled cells in the VZ (arrowheads) in cL regions of E12.5 ablated telencephalons with respect to control littermates. No defects in differentiation were observed in rD regions at this stage () correlating with the presence of numerous Dbx1
-derived CP transient neurons in cL but not in rD territories at E12.5 (Teissier et al. 2010
). Interestingly, by E14.5, a similar increase in differentiation was observed in the rD region of mutant telencephalons ( and data not shown), whereas no differences were detected in the CP of the cL region, with the exception of ectopic Tbr1+
neurons in the VZ (, arrowheads). Moreover, at E14.5 a slight thinning of the VZ was apparent especially in mutant cL territories (, black bars).
Figure 2. Transient increase in differentiation progressively affects the entire pallium of E1-Ngn2/CRE;Dbx1DTA mutants. (A–H) Immunohistochemistry performed on E12.5 embryos shows no differences for Tbr1 (A,B) and TuJ1 (C,D) staining between control ( (more ...)
In order to assess if the increase in differentiation was due to defects in proliferation occurring upon ablation, we quantified the number of mitotic cells, as labeled by PH3 staining, in the rD and cL regions of the developing pallium at E12.5, E14.5, and E16.5 (). We detected an initial increase in the number of PH3+ mitotic progenitors in the cL VZ at E12.5 followed by a reduction clearly observed at E16.5 (). At E14.5, corresponding to the time of arrival of the CP transient neurons in rD territories in control animals, enhanced VZ proliferation was observed in rD regions of mutant cortices (), followed by a small decrease by E16.5 (). Notably, the number of PH3+ mitotic progenitors in the SVZ was strongly reduced at both cL and rD levels starting at E14.5 ().
Taken together, these results show that the loss of the CP transient neurons leads initially to an increment in the number of mitosis in the VZ and of differentiating neurons (at E12.5 in cL and E14.5 in rD regions), followed by a decrement in the number of both VZ and SVZ mitosis. Notably, changes in proliferation and differentiation appear to progress from the caudolateral to the rostrodorsal cortex and to correlate with the timing and trajectory of invasion of pallial regions by the CP transient neurons.
CP Transient Neurons Ablation Results in Precocious Neurogenesis
The defects observed in ablated animals could result from either a change in cell cycle length, an increase in the fraction of neurogenic divisions and/or a variation in the size of the progenitor pools. In order to discriminate between these possibilities, we started by measuring the length of the cell cycle using IdU and BrdU injections 3 and 1.5 h before collection of the embryos (see Materials and Methods) (). At E12.5, we observed lengthening of the cell cycle in progenitors located in the VZ in cL (Tc = 13.31 h ± 2.91 in controls and Tc = 25.71 h ± 4.02 in mutants) but not in rD territories (Tc = 9.19 h ± 1.07 in controls and Tc = 11.96 h ± 1.09 in mutants). By E14.5, VZ progenitors at both cL and rD levels displayed a clear increase in Tc (rD: Tc = 11.74 h ± 0.54 in controls and Tc = 18.51 h ± 3.27 in mutants; cL: Tc = 17.81 h ± 2.80 in controls and Tc = 27.96 h ± 3.91 in mutants). Therefore, defects in proliferation and differentiation correlate with a lengthening of the cell cycle in E1-Ngn2/Cre;Dbx1DTA mutant embryos.
Figure 4. Elongated cell cycle length and enhanced neurogenic divisions in progenitors upon ablation of the CP transient neurons. (A) Schematic illustration of the double S-phase–labeling paradigm for the estimation of cell cycle length. (B) Graphs show (more ...)
A lengthening of the cell cycle has been shown to characterize differentiative divisions (Miyama et al. 1997
; Calegari et al. 2005
). To address whether an increased fraction of cells exited the cell cycle (Q fraction) in E14.5 E1-Ngn2/Cre;Dbx1DTA
animals, we performed a single injection of IdU followed by multiple injections of BrdU (every 2 h) for a total of 15 h () and quantified the number of cells which failed to reentered the S-phase during the time of the experiment (IdU+
) among actively cycling cells (IdU+
) (see Materials and Methods). We observed that an increased proportion of cells exited the cell cycle in ablated cortices compared with controls at both cL and rD levels (). Evaluation of the total number of postmitotic neurons generated during one cell cycle by counting the number of Tbr1+
cells relative to the size of the progenitor pool (IdU+
) also confirmed an enhanced fraction of neurogenic divisions in mutant compared with control littermates (). Furthermore, we detected an increase in the expression of Ngn2
, a proneural gene detected at high levels in neurogenic divisions (Shimojo et al. 2008
; Ochiai et al. 2009
), in the VZ and SVZ of mutant compared with control littermates (). Together, these experiments suggested that there was an augmentation in the number of progenitors undergoing neurogenic instead of proliferative divisions in both the VZ and SVZ of ablated embryos.
To further analyze whether the cortical neuroepithelium was undergoing precocious neurogenesis, we performed coimmunostaining for Tbr2, a gene expressed in both IP cells and early postmitotic neurons in the developing pallium (Englund et al. 2005
) and TuJ1. We observed an increase in the number of TuJ1+
cells in the VZ of E14.5 E1-Ngn2/Cre(iresGFP);Dbx1DTA
mutant embryos and especially in that of TuJ1+
cells in the VZ (green arrowheads in ). We also noticed that ectopic Tbr2+
cells were detected in the apical VZ almost at the ventricle in mutant animals (red arrowheads in ). Notably, a general increase in expression of the GFP, corresponding to the activity of the E1
enhancer element of the Ngn2
gene, was also detected in mutants particularly at the apical VZ () and correlated with the enhanced Ngn2
staining previously described. All GFP+
cells were Tbr2+
in the VZ, but not reciprocally, in both control and mutant E1-Ngn2/Cre(iresGFP);Dbx1DTA
cortices (), suggesting that the GFP labels a specific subpopulation of the Tbr2+
cells. Moreover, the TuJ1+
cells in the apical VZ observed in control as well as the ectopic ones observed in mutant animals were almost always associated with GFP expression (green arrowheads in ), suggesting that the Tbr2+
cells in the apical VZ are postmitotic neurons. Accordingly, we observed a higher proportion of Tbr2+
cells among Tbr2+
cells at E12.5 in cL territories (cL: 20.62 ± 2.65% in controls compared with 51.42 ± 2.96% in mutants, the GFP is not expressed at rD level at this stage). A similar increase was detected in both cL and rD regions at E14.5 (cL: 31.85 ± 1.79% in controls compared with 52.76 ± 0.18% in mutants; rD: 29.39 ± 11.79% in controls compared with 63.19 ± 3.03% in mutants) and maintained at E16.5 (cL: 15.06 ± 1.40% in controls compared with 45.82 ± 9.68% in mutants; rD: 15.75 ± 0.78% in controls compared with 24.67 ± 0.46% in mutants). We conclude that an enhanced fraction of Tbr2+
cells corresponds to postmitotic neurons upon ablation of the CP transient neurons.
Thus, together these experiments demonstrate that progenitors throughout the neocortical neuroepithelium undergo precocious neurogenic divisions in E1-Ngn2/CRE;Dbx1DTA animals by midneurogenesis.
Depletion of Neocortical Progenitor Pools in Ablated Mutants
An increase in the neurogenic fraction of progenitor divisions and a decrease in that of proliferative divisions should lead to a progressive depletion of the progenitor pool. Indeed, the observed decrease in the number of mitosis in E14.5 and E16.5 in E1-Ngn2/Cre;Dbx1DTA embryos together with a reduction in Ki67 staining, a marker of proliferative cells, at caudal levels of E14.5 mutant pallium compared with control littermates () strongly suggested a global depletion of the progenitor pools.
Since the decrease in the proliferation was most pronounced in the SVZ at E14.5, we first tested whether the pool of IPs was affected. We quantified the number of Tbr2+ cells within one column spanning the thickness of the developing pallium and observed a reduction starting in the cL region of mutant telencephalons at E12.5 (). Strong defects were observed in rD territories at E14.5 and E16.5 but not at E12.5. Therefore, together with the enhanced number of postmitotic Tbr1+ and Tbr2+ neurons at E12.5 and E14.5 in cL and rD regions, respectively () and the reduction in SVZ proliferation (), these results show that the decrease in the total number of Tbr2+ cells corresponds to a depletion in the pool of IPs rather than of early postmitotic neurons (also Tbr2+).
In order to determine if the pool of RGs was also affected, we analyzed the expression of BLBP and Pax6 proteins, 2 specific markers of RG cells, at E16.5. We observed a decrease in their expression in mutant cL cortices () correlating with a ~25% reduction in cell density, as measured by DAPI staining, in the VZ (). In addition, we observed at E16.5 a decrease in BrdU labeling (upon a 2 h pulse) in the VZ and SVZ of both rD and cL regions (). We conclude that a depletion of both the IP and the RG progenitors' pools occurs in the pallium of the E1-Ngn2/Cre;Dbx1DTA animals.
Increased Number of Dbx1-Derived CP Transient Neurons in the Pallium of Gsx2 Mutants
Since the E1-Ngn2/CRE;Dbx1DTA
mutants represent a specific ablation of Dbx1
-expressing progenitors at the VP, we sought to analyze Gsx2
null mice which have been reported to display opposite effects, namely an expansion of the Dbx1
expression domain in the VP (Yun et al. 2001
; Carney et al. 2009
). Notably, these mutants have recently been shown to present an increase in the number of Dbx1
-derived glutamatergic neurons migrating into the lateral amygdala (Waclaw et al. 2010
). The role of Gsx2
in dorsoventral patterning and neurogenesis of the subpallium has been analyzed in several studies (Corbin et al. 2000
; Yun et al. 2001
; Stenman, Toresson, et al. 2003
; Yun et al. 2003
; Carney et al. 2009
). However, no major defects in pallial development had been reported so far.
In order to permanently trace Dbx1
-derived CP transient neurons in the pallium of the Gsx2
mutants, we crossed Dbx1CRE/+;Gsx2RA/+
animals (Waclaw et al. 2010
). By immunostaining for Tbr1 on Dbx1CRE/+;CagCat-EGFP;Gsx2+/+
cortices, we observed an increased stream of Dbx1
-derived glutamatergic neurons (GFP+
) migrating dorsally into the pallium of E13.5 homozygous mutants () compared with control littermates (). In addition, quantification of the number of GFP+
cells coexpressing Tbr1 and/or Mef2c, a gene which labels VP-derived neurons migrating into the amygdala (Waclaw et al. 2010
), revealed a 124 ± 3.18% and 274.66 ± 5.72% increase in the CP of E18.5 Gsx2
mutants (). Most supernumerary Dbx1
-derived neurons were positioned in deep layers as expected from their early birthdate. No significant differences in the number or location of Dbx1
CR cells was observed in E13.5 and E18.5 Dbx1CRE/+;CagCat-EGFP;Gsx2+/+
cortices (data not shown and ). We conclude that Gsx2
mutant animals display a specific increase in the number of CP transient neurons migrating into the pallium from early stages of development.
Figure 6. Increased number of Dbx1-derived CP transient neurons migrating in the developing pallium of Gsx2 mutants. (A–D) Immunohistochemistry for GFP and Tbr1 shows an increase in the number of GFP+ cells migrating tangentially into the pallium of E13.5 (more ...)
Enhanced Proliferation and Neurogenesis in the Pallium of Gsx2 Mutants
We started by analyzing the progression of differentiation using Tbr1 and TuJ1 immunostaining in E12.5 Gsx2
mutants and observed a global decline affecting both cL and rD regions, but this was more pronounced at cL levels (Supplementary Fig. S2
). We also detected a concomitant enhanced proliferation in both the VZ and the SVZ, as revealed by Ki67 and BrdU staining (Supplementary Fig. S2
). At E14.5, a strong increment in Ki67 and PH3 immunostaining was detected in both the VZ and the SVZ of Gsx2
mutants at cL and rD levels ( and data not shown). Quantification of PH3+
cells at the apical VZ and the SVZ confirmed a higher number of ventricular and abventricular mitosis in these mutants (). In addition, we observed more numerous Tbr2+
cells in the SVZ () but not at the apical VZ (), showing that both RG and IP progenitors display enhanced proliferation. Finally, we observed a reduction in Ngn2
staining in both the VZ and the SVZ of mutant animals () as well as a reproducible slight decrease in Tis21
expression, especially observed in the apical portion of the VZ (), strongly arguing for a decrease in the fraction of neurogenic divisions. Notably, the number of Tbr1+
neurons in the CP of E14.5 Gsx2
mutants was similar to that of control embryos (data not shown). Therefore, together these results suggest that an amplification of both RGs and IPs progenitor pools and a delayed neurogenesis occur in the pallium of Gsx2
mutants. These defects are opposite with respect to the precocious differentiation and depletion of the progenitor pools described in the E1-Ngn2/CRE;Dbx1DTA
mutants at corresponding stages.
We then analyzed the phenotype at E18.5, the latest stage possible for Gsx2 mutants which die at birth and observed an increase in the thickness of the CP at both rD (25.49%) and cL (30.96%) levels. Tbr1 and Cux1/2 immunostaining showed that both deep and superficial layers were thicker () and appeared to be affected in a similar manner (), although the supernumerary CP transient neurons were positioned in deep layers. Finally, we observed a strong increase of neuronal numbers in layers V and II–IV upon immunostaining for FoxP2, Ctip2 and Mef2C, as well as by in situ hybridization for Cdh8 (). In conclusion, we have shown that Gsx2 mutants present a surplus number of CP transient neurons and display opposite non-cell autonomous pallial defects to those observed upon their ablation in E1-Ngn2/CRE;Dbx1DTA animals, namely excessive proliferation of both VZ and SVZ progenitors and a consequent increased number of cortical neurons fated to both deep and superficial layers.