Subcerebral projection neurons are absent from the cortex of Fezf2−/− mice and are substituted by deep layer callosal projection neurons
Loss of the transcription factor Fezf2
results in the absence from the cortex of all subcerebral projection neurons, which fail to develop while other projection neuron types across all cortical layers are generated normally (Chen et al., 2005a
; Chen et al., 2005b
; Molyneaux et al., 2005
). Loss of subcerebral neurons in this mutant is accompanied by the expansion of a different population of projection neurons with callosal projection neuron properties (Chen et al., 2008
) without overall loss of neurons, defective migration or changes in cortical thickness (Molyneaux et al., 2005
). Despite Fezf2−/−
mice are viable and motile (Molyneaux et al., 2005
), they are prone to develop seizures upon handling or exposure to auditory stimuli (unpublished data).
To determine whether the abnormal development of projection neurons in this mutant affects the distribution of cortical interneurons and whether specific subtypes of projection neurons play different roles, we first defined the nature of the CPN that replace subcerebral projection neurons in the Fezf2−/−
cortex. As expected, CTIP2, a marker of subcerebral projection neurons was not expressed in mutant layer V (Molyneaux et al., 2005
). In contrast, TBR1, a marker of layer VI and layer II/III neurons, and SATB2, a protein important for CPN development, were both expanded (Chen et al., 2008
; Molyneaux et al., 2005
) and were co-expressed in a new population of projection neurons located in layer Vb (Figure S1G–P
). To determine whether these neurons project through the corpus callosum, we retrogradely labeled CPN of the mutant mice with FluoroGold (FG) injections in contralateral hemisphere. Despite the fact that most CPN form Probst bundles (Pb), making their retrograde labeling inefficient, FG-labeled CPN were visible within layer V and co-expressed TBR1 and SATB2 (). These data extend prior electrophysiological analysis and characterization of these neurons in chimeric mice by demonstrating that they project through the corpus callosum (Chen et al., 2008
). These CPN are distinct from the CPN of layer Va, which expressed SATB2 but not TBR1 (data not shown). To precisely define the subtype-specific identity of CPN in the mutant layer V, we analyzed the expression of three CPN-specific genes among several that were shown to label subpopulations of CPN in different layers (Molyneaux et al., 2009
labels CPN in layers V, VI and in the deeper part of layer II/III; Inhba
labels CPN of layer II/III; and Limch1
is expressed in subpopulations of CPN within the upper part of layer II/III. In the Fezf2−/−
expression was increased in layer V (; arrows), whereas expression of Inhba
remained unchanged (; arrowheads). This molecular and hodological analysis indicates that CPN from layers Vb/VI, but not from layers Va or II/III, replace subcerebral projection neurons within layer V of the Fezf2−/−
Neurons that replace subcerebral projection neurons in Fezf2−/− cortex are CPN of deep layer identity
GABAergic interneurons acquire abnormal radial distribution in the Fezf2−/− cortex
Fezf2−/− mice represent a unique mutant model in which a single projection neuron population fails to develop and is substituted by projection neurons of a different identity. This motivated us to use this mice to determine whether projection neurons control the laminar distribution of GABAergic interneurons, and, further, whether different types of projection neurons have different roles. (). First, we quantified the number of GAD67-positive interneurons detected by in situ hybridization in motor, somatosensory and visual cortex at P28 in wild type and Fezf2−/− littermates (n=3 wt; n=3 Fezf2−/−). The total number of GAD67-positive interneurons did not differ between wild type and mutant corteces (), but the distribution of GAD67 interneurons was distinctly abnormal in the Fezf2−/− mutant ().
The Fezf2−/− cortex has abnormal radial distribution of GABAergic interneurons
To precisely determine the radial distribution of GAD67-positive interneurons, we divided the cortex into 10 bins of equal size spanning the cortical thickness. Calculation of the percentage of interneurons located in each bin demonstrated a clear reduction of interneurons within lower bins across areas of the Fezf2−/− cortex. This difference was particularly prominent for bins 4–5 in the motor and somatosensory areas, and bins 3–4 in the visual area, and it was accompanied by an increase of interneuron percentages in more superficial bins (). Double staining for GAD67 and β-galactosidase (labeling subcerebral projection neurons) in Fezf2+/− mice showed that bins with reduced percentages of interneurons mostly corresponded to layer V (). This anatomical information on layer positioning allowed us to assign bins to three groups spanning layers VI, V and II–III/IV. Analysis of interneuron distribution within each of these layers highlighted acute phenotypic abnormalities within layer V (). Reduced percentages of interneurons in Fezf2−/− layer V were observed across all cortical areas sampled. This was accompanied by increased interneuron percentages in the Fezf2−/− superficial layers II–III/IV in somatosensory and visual areas (with a similar trend in the motor area). In contrast, layer VI was unaffected ().
To understand whether the reduced number of interneurons in layer V of the Fezf2−/−
cortex might reflect a smaller number of interneurons normally associated with CPN compared to subcerebral projection neurons, we quantified the number of GABA-positive interneurons surrounding these two projection neuron populations within wild type layer V. We found significantly fewer interneurons associated with SATB2-expressing CPN in layer Va than with CTIP2-positive subcerebral projection neurons in layer Vb (n=3; p value=0.001) (Figure S2
). This suggests that the reduced percentages of interneurons observed in layer V of the mutant cortex is in line with a typically lower number of cortical interneurons distributed around deep layer CPN.
Together, these data indicate that upon reaching the cortex, interneurons require projection neurons in order to acquire proper lamination. Furthermore, given that in the Fezf2−/− cortex there is a precise replacement of subcerebral projection neurons with CPN, these findings demonstrate that different types of excitatory projection neurons differentially affect the distribution of cortical interneurons.
Fezf2−/− mice exhibit unbalanced cortical activity due to defective GABAergic inhibition
To determine whether the observed changes in interneuron distribution results in unbalanced cortical activity and physiology, we used voltage-sensitive dye imaging (VSDI) (Grinvald and Hildesheim, 2004
) to examine spatio-temporal dynamics of functional connections in the Fezf2−/−
mice. The spread of activity through coronal slices of the visual cortex in response to a current pulse delivered to the white matter was quantified. As expected, wild type and Fezf2+/−
mice exhibited a strong response that propagated rapidly to the upper layers “on beam” with the stimulating electrode, spreading only weakly along the deep layers even at threshold stimulating strengths ( and Movie S1
). In contrast, the response in Fezf2−/−
slices at threshold rarely reached the upper layers, and remained largely confined to the lower layers ( and Movie S2
Spread of neuronal activity is restricted to the deep layers in Fezf2−/− cortex
Maximum fluorescence intensity was quantified within two 125 mm2 regions in-line with the stimulating electrode, one in upper layers (; black box) and one in lower layers (; white box). Input-output curves revealed an increase in upper layer response with increasing stimulus intensity, however the response failed to reach wild-type levels in the upper layers of Fezf2−/− mice across all stimuli (). Conversely, lower layer responses were consistently stronger in Fezf2−/− mice compared to wild type (). These differences were significant for both upper and lower layers at half maximal stimulation ().
Physiological imbalance of excitation across cortical layers in Fezf2−/−
cortex may be explained by abnormal excitatory networks, altered inhibitory GABAergic interneurons, or both. To distinguish among these possibilities, we performed VSDI measurements in the presence of the GABAA
receptor antagonist bicuculline. Strikingly, the laminar differences between genotypes were eliminated under these conditions (Movies S3
), indicating that the excitatory network scaffold is intact in the Fezf2−/−
mutant cortex. Taken together, an increased GABAergic tone in the superficial layers of the mutant cortex (without defects in excitatory network function) provides physiological support for our histological findings that interneuron numbers are reduced in layer V and increased in the upper layers II–III/IV of the Fezf2−/−
Fezf2 does not affect the fate specification of cortical interneurons
Defects of interneuron fate specification or tangential migration could account for abnormal lamination of interneurons. Therefore, we investigated whether Fezf2
plays a cell-autonomous role in the fate specification or migration of GABAergic interneurons in the ventral forebrain. We have previously reported that with the exception of a small area in the developing amygdala, Fezf2
is absent from the entire ventral telencephalon, and is not expressed in the MGE and CGE, where cortical GABAergic interneurons are born (Rouaux and Arlotta, 2010). In the dorsal telencephalon, Fezf2
is expressed in progenitors, and within the cortical plate at high levels in subcerebral projection neurons and at low levels in layer VI projection neurons (Chen et al., 2005a
; Chen et al., 2005b
; Molyneaux et al., 2005
). Here, we found that Fezf2
was also not expressed in interneurons within the cortex, as assessed by crossing GAD67::GFP mice with Fezf2−/+
mice that carry a LacZ
reporter gene at the Fezf2
locus. GFP-positive interneurons did not colocalize with β-galactosidase- and CTIP2-positive subcerebral neurons (Figure S3A–F
which normally control the specification of early-born interneuron progenitors in the MGE, and Lhx6,
which labels their postmitotic interneuronal progeny, were unchanged in the mutant (Figure S3G–L
). Similarly, COUPTF-II,
which labels interneuron progenitors within the CGE displayed comparable expression in wild type and mutant mice (Figure S3M,N
These data demonstrate that in the telencephalon Fezf2 expression is excluded from GABAergic interneurons and does not affect expression of genes that are critical for interneuron fate specification.
Subcerebral projection neurons are required for the proper distribution of SST- and PV-expressing interneurons, but not CR-expressing interneurons
Despite the fact that cortical interneurons are heterogeneous and it is not currently possible to precisely associate specific interneuron subtypes with strict layer locations, it is known that early-born, SST- and PV-expressing interneurons preferentially populate the deep layers of the cortex (Butt et al., 2005
; Cobos et al., 2006
; Fogarty et al., 2007
), while mostly late-born, CR-expressing interneurons are present in higher numbers in the superficial layers (Miyoshi et al., 2010
; Nery et al., 2002
; Xu et al., 2004
). To define whether the absence of subcerebral projection neurons affects the distribution of all interneurons equally or exerts selective control over interneuronal populations that normally occupy the same deep layers of the cortex, we investigated the distribution of SST-, PV-, and CR-expressing interneurons in the Fezf2−/−
Given that the distribution of GAD67-positive interneurons was abnormal in layer V across all areas sampled, we concentrated our analysis on one representative area: the somatosensory cortex. First, we studied the distribution of SST-expressing interneurons, which in wild type cortex are present in high numbers within layer V () (P28; n=3 wt; n=3 Fezf2−/−). We found that the total number of SST-expressing neurons was unchanged in the mutant cortex compared to wild type (). However, the typical laminar distribution of SST interneurons was strikingly altered in the mutant (), with clear decreased percentages in layer V (bins 4–5) and increased percentages in layers II–III/IV (bins 6–10) ().
SST- and PV-interneuron subtypes are reduced in layer V and increased in the superficial layers of the Fezf2−/− cortex
PV-expressing interneurons also showed comparable, abnormal radial distribution (n=3 wt; n=3 Fezf2−/−
). The percentage of PV-expressing interneurons in the somatosensory cortex at P28 was decreased in layer V and increased in the upper layers II–III/IV, without a change in the total number of PV-positive interneurons (). In stark contrast to the abnormal distribution of SST
- and PV-expressing interneurons, CR-expressing interneurons were not affected by the absence of subcerebral projection neurons and were distributed normally in the P28 Fezf2−/−
cortex (n=3 wt; n=3 Fezf2−/−
) (Figure S4A–F
These findings strongly suggest that distinct projection neuron subpopulations selectively and preferentially affect the distribution of cortical interneuron subtypes that are normally destined to populate the same cortical layers.
Interneuron reduction in layer V of Fezf2−/− cortex is not due to abnormal connectivity by CPN or interneuron cell death
The reduced percentages of cortical interneurons in Fezf2−/−
layer V suggest that CPN affect interneurons differently from the subcerebral projection neurons that they replace. However, it is possible that the presence of Probst bundles (Pb) and thus the altered connectivity by CPN across the corpus callosum may have affected their ability to interact with interneurons in the Fezf2−/−
cortex. In order to understand whether development of Pb per se
could cause the observed abnormalities, we analyzed the distribution of SST
-expressing interneurons in SV129S1/SvImJ wild type mice, a strain where females sporadically develop Pb without changes in other cerebral commisures (Wahlsten et al., 2003
and data not shown). The total number and dorso-ventral binned distribution of SST
-expressing interneurons in somatosensory areas of SV129S1/SvImJ female mice with Pb compared to female littermates with a corpus callosum (cc), showed no differences (total interneuron numbers, Pb mice: 125.93+/−6.23, n=3; control mice: 135.08+/−10.96, n=3; p value= 0.51) (Figure S5A–E
). This strongly suggests that abnormal connectivity by CPN to contralateral targets is not sufficient to influence interneuron layer positioning. In addition, our VSDI data show that projection neurons in the mutant cortex were excitable, a finding supported by prior electrophysiological recordings (Chen et al., 2008
Increased interneuron cell death could have also accounted for the reduced percentage of interneurons in Fezf2−/−
layer V (Cobos et al., 2006
). To investigate this possibility, we performed staining for FluoroJade-C, which broadly labels dying neurons, in wild type (n=3) and Fezf2−/−
(n=3) cortex at P0, P7 and P14, by which time interneuronal abnormalities are distinctly evident (data not shown). There was no difference in the number of FluoroJade-C positive neurons in wild type and mutant somatosensory areas at any of the ages sampled (Figure S5F–J
). In addition, we did not detect any increase in Caspase-3 staining in GAD67
-expressing interneurons (Figure S5K–N
). Together with the finding that the total number of interneurons is unchanged in the Fezf2−/−
cortex at P28, these data demonstrate that increased cell death did not account for the abnormal distribution of cortical interneurons in this mutant.
De novo generation of corticofugal projection neurons is sufficient to recruit deep layer cortical interneurons to ectopic locations
Our loss-of-function data demonstrate that a fate switch between two populations of projection neurons leads to a change in the distribution of specific subtypes of interneurons. This suggests that signaling between projection neuron and interneuron subtypes may be a mechanism by which projection neuron classes instruct the recruitment of specific interneuron partners.
To investigate this possibility, we overexpressed Fezf2
by in utero
electroporation in cortical progenitors at E14.5, a stage at which projection neurons of the superficial layers, mostly CPN, are generated. Elevated levels of Fezf2
in these late-stage neural progenitors instruct the heterochronic generation of virtually pure corticofugal projection neurons (including subcerebral projection neurons). As previously reported, Fezf2
-induced corticofugal neurons did not migrate into the cortex, rather they developed in ectopic clusters below the corpus callosum and extended corticofugal axonal projections to the thalamus and the pons (Figure S6C
, arrow) (Molyneaux et al., 2005
). They also expressed TBR1, TLE4 and SOX5, markers of corticofugal projection neurons and CTIP2, which is expressed at high levels in subcerebral projection neurons (Figure S6B–E,G–P
). None of the Fezf2
-induced neurons expressed SATB2 or CUX1, molecular markers of CPN (Figure S6D,F,G,Q–S
). Therefore, overexpression of Fezf2
results in the generation of a new population of corticofugal projection neurons outside of (yet in close proximity to) the cortex.
We examined expression of GABA in these ectopic corticofugal neuron clusters to determine if interneurons might be present within them. In contrast to contralateral matched locations, we found that many GABAergic interneurons invaded these aggregates (). This finding was confirmed using in situ
hybridization to detect ectopic GAD67
-positive cells (). We quantified the number of GABA-positive interneurons within each aggregate and normalized it to the area covered by the GFP-positive corticofugal neurons. Interestingly, the number of interneurons present directly correlated with the size of the aggregate (Figure S7
), suggesting that projection neurons recruit cortical interneurons in proportion to their own numbers.
Experimentally-generated aggregates of corticofugal projection neurons recruit cortical interneurons to new ectopic locations
Strikingly, ectopic corticofugal projection neurons appear to play an attractive role upon cortical interneurons in an in vitro
migration assay (Figure S8
was electroporated in utero
in cortical progenitors at E14.5 as described above, and the induced clusters of corticofugal neurons were microdissected at E18.5 and cultured in proximity to explants of MGE from Lhx6
-GFP positive, E12.5 embryos (Figure S8D
). As controls, we cultured E12.5 MGE explants alone (Figure S8A
). As expected, control MGE explants displayed unbiased outgrowth of GFP-positive interneurons in all directions around the explant (Figure S8B,C
). In contrast, interneurons showed polarized migration towards the cluster of Fezf2
-expressing corticofugal neurons (Figure S8E,F
). This indicates that experimentally generated corticofugal neurons can attract cortical interneurons, a finding in agreement with the demonstration that cortical plate neurons attract interneurons in a similar in vitro
assay (Lopez-Bendito et al., 2008
We then asked whether the ectopic clusters of corticofugal neurons recruit interneurons that are characteristic of the deep layers of the cortex. Remarkably, many Lhx6-
positive and SST-
positive interneurons, which normally populate primarily the deep cortical layers, were found within the corticofugal neuron aggregates (). In contrast, NPY-expressing interneurons, normally restricted to the superficial layers were not present in these clusters (). Similarly, VIP
-expressing interneurons, which show a less restricted, yet still preferential distribution in the upper layers, were only present in very low numbers, likely corresponding to those normally located in the deep layers (). Remarkably, Reelin, a gene that among interneurons labels those located in layers II/III and Va, (Alcantara et al., 1998
) was absent from the corticofugal neuron aggregates ().
Interneurons characteristic of deep-layers are selectively found in corticofugal neuron aggregates
Together with our loss-of-function data, these gain-of-function results support a model by which deep layer corticofugal projection neurons selectively affect the distribution of interneuron subtypes that normally localize to the same layers.
Ectopically positioned upper layer II/III callosal projection neurons can recruit cortical interneurons
The ability to recruit interneurons could be a unique property of deep layer corticofugal projection neurons or might be applicable, more broadly, to other types of projection neurons. To investigate this possibility directly, we experimentally induced ectopic clusters of layer II/III callosal neurons and tested their ability to affect interneuron positioning. It has previously been shown that knock-down of the β-Amyloid Precursor Protein (APP
) gene in rat cortical progenitors results in arrested migration of projection neurons (Young-Pearse et al., 2007
). We took advantage of this system to focally arrest layer II/III CPN below the corpus callosum ().
Ectopically-located aggregates of layer II/III callosal projection neurons recruit cortical interneurons
A construct carrying an APP shRNA (referred to as U6-APP shRNA-2) was electroporated in utero together with a reporter CAG-GFP construct at E14.5, when upper layer CPN are generated. Mice were sacrificed at P6, by which time distinct clusters of neurons were visible below the corpus callosum (). The location of these clusters corresponded precisely to that of Fezf2-induced corticofugal neuron aggregates. As expected, the ectopic neurons expressed SATB2 and CUX1, and did not express CTIP2 ( and data not shown), confirming that they differentiated appropriately into upper layer CPN. GABA immunocytochemistry on these CPN clusters (highlighted by the expression of SATB2) showed the distinct presence of GABA-positive interneurons within the aggregates, compared to matched positions in the contralateral hemisphere (n=9; ). The experiment was also performed in the rat with identical results (n=3; data not shown).
These results demonstrate that layer II/III CPN are also able to recruit interneurons and further support a model by which projection neurons are generally able to affect interneuron lamination.
Projection neurons recruit interneurons that are appropriate for their projection neuron subtype-specific identity and not strictly their birthdate
It is intriguing that cortical projection neurons and interneurons that are synchronically born preferentially populate the same cortical layers (Fairen et al., 1986
; Miller, 1985
; Peduzzi, 1988
; Pla et al., 2006
; Valcanis and Tan, 2003
). Early-born (peak at E13), largely MGE-derived interneurons localize in deep layers, whereas late-born (peak at E15), largely CGE-derived interneurons occupy the superficial layers (Fairen et al., 1986
; Peduzzi, 1988
; Pla et al., 2006
). Our finding that ectopic corticofugal projection neurons specifically recruit large numbers of Lhx6
- and SST
-expressing deep layer interneurons despite being born synchronically with superficial layer CPN led us to investigate whether the projection neuron-type identity, rather than strictly the projection neuron birth date, affects the choice of interneurons recruited.
We examined whether the Fezf2-induced corticofugal projection neurons heterochronically generated two days after the bulk production of endogeneous corticofugal neurons has ended preferentially recruit interneurons that are appropriate for their projection neuron-type identity (i.e. interneurons born at E12–E13) or, rather, interneurons appropriate for their heterochronic day of birth (i.e. interneurons born the day of the electroporation, at E14.5). In complementary experiments, we investigated whether upper layer II/III CPN that were synchronically born with Fezf2-induced corticofugal neurons at E14.5 and that were similarly positioned below the corpus callosum (due to APP knock-down), recruited interneurons born at the same time.
In separate animals, we administered BrdU to timed-pregnant females at either E12.5 or E14.5, combined with either Fezf2 overexpression or APP downregulation in cortical progenitors at E14.5 (). Mice were sacrificed at P6. Quantification of BrdU-labeled, GABA-positive interneurons showed that heterochronic, late-born corticofugal projection neurons preferentially recruited interneurons that were born early, at E12.5 (). These are appropriate for the deep-layer identity of the corticofugal projection neurons generated, but not for their heterochronic experimental birthdate (E14.5). In agreement with the E14.5 experimental birthdate of the corticofugal projection neurons, neuronal aggregates contained high numbers of E14.5 Brdu-labeled, GABA-negative projection neurons (). Confirming the specificity of this effect, we found that upper layer II/III CPN populations born the same day as the experimentally generated corticofugal neurons specifically attracted late-born interneurons (). This is noteworthy, since in this experimental system, corticofugal neurons and layer II/III CPN shared the same day of birth and were similarly located below the cortex, giving them access to comparable pools of cortical interneurons.
Ectopic aggregates of corticofugal projection neurons recruit interneurons that are appropriate for their projection neuron-type identity
To determine whether the relative distribution of early- and late-born interneurons found in these aggregates corresponded to the distribution normally present in the deep or superficial layers of the cortex, we quantified the number of E12.5- and E14.5-born interneurons within the deep and superficial layers overlying the aggregates. Strikingly, Fezf2-corticofugal aggregates had similar relative proportions of early- and late-born interneurons as found in the deep layers of the cortex (). In a complementary trend, APP shRNA-layer II/III CPN showed the same distribution of early- and late-born interneurons as present in the superficial layers (). We conclude that cortical projection neurons select interneuron partners that are appropriate for their specific projection neuron identity and not strictly based on synchronic birthdates.