Ephrin-A2 regulates excitatory neuron differentiation and interneuron migration in the developing neocortex

The development of the neocortex requires co-ordination between proliferation and differentiation, as well as the precise orchestration of neuronal migration. Eph/ephrin signaling is crucial in guiding neurons and their projections during embryonic development. In adult ephrin-A2 knockout mice we consistently observed focal patches of disorganized neocortical laminar architecture, ranging in severity from reduced neuronal density to a complete lack of neurons. Loss of ephrin-A2 in the pre-optic area of the diencephalon reduced the migration of neocortex-bound interneurons from this region. Furthermore, ephrin-A2 participates in the creation of excitatory neurons by inhibiting apical progenitor proliferation in the ventricular zone, with the disruption of ephrin-A2 signaling in these cells recapitulating the abnormal neocortex observed in the knockout. The disturbance to the architecture of the neocortex observed following deletion of ephrin-A2 signaling shares many similarities with defects found in the neocortex of children diagnosed with autism spectrum disorder.

triggering bi-directional responses: a forward signaling cascade in the receptor-bearing cell and a reverse signaling cascade in the ligand-bearing cell [21][22][23] . Receptor-ligand interactions principally elicits repulsion, contributing to the migration and segregation of neuronal populations including neocortical excitatory neurons 24,25 and interneurons [26][27][28] . Eph/ephrin interactions also regulate other aspects of neocorticogenesis, including brain size through regulation of apoptosis 29 and progenitor proliferation 30 .
Here we describe focal lamination defects in the neocortex of all adult ephrin-A2 homozygote knockout mice (efnA2 KO), forming discrete patches of reduced neuronal density. Their distribution is disparate and not restricted to specific neocortical areas, highly reminiscent of similar aberrations observed in the neocortex of children diagnosed with Autism Spectrum Disorder (ASD) 31 . Using in vivo and ex vivo knockdown approaches we demonstrate that ephrin-A2 contributes to the establishment of excitatory and inhibitory neocortical neurons, with distinct roles in each population. Specifically, ephrin-A2 promotes the migration of interneurons emerging from the POA and modulates the proliferation of apical progenitors in the neocortex. We propose that ephrin-A2 acts as a proneural cell fate determinant, with the loss of ephrin-A2 resulting in abnormal neocortical lamination akin to ASD-associated deficits.

Results
Disruption of the neuronal cytoarchitecture in the neocortex of efnA2 KO mice. The neocortical cytoarchitecture of adult mice lacking efnA2 appeared normal when labeled with the DNA stain, Hoechst (Fig. 1a). However, labeling with the neuronal marker, NeuN, revealed discrete patches with reduced density of neuronal cell bodies (Fig. 1a') and dendritic arborisation (Fig. S1a,b'). The patches established sharp boundaries between normal and disrupted zones and extended through the neocortex with the loss of neurons becoming more severe towards the center of the patch, as illustrated on the series of adjacent sections (Fig. 1a'). The phenotype varied in severity, yet occurred with a 100% penetrance, with all mutant brains exhibiting at least one disorganized patch per hemisphere. Their distribution, however, was highly variable and apparently random, with the disruption affecting all layers indiscriminately (Fig. 1b' , layers 2/3; Fig. 1c' , layer 5), confirmed by a signal reduction on the fluorescence intensity profile (Fig. 1b", c"). The stability of the Hoechst nuclear labeling (Fig. 1b", c") and haematoxylin and eosin histology (Fig. S1c, d') demonstrates that despite the reduction in the density of NeuN+ cells, the neocortical patches were uniformly populated with structurally normal cells. To characterize the identity of the cells localized within the patches we double-labeled efnA2 KO sections with NeuN to demarcate the abnormal regions, and either ALDH1L1, Olig2, or Iba1 to label astrocytes, oligodendroglia or microglia/macrophages respectively. Both astrocytes and oligodendrocytes were noticeably reduced within the regions lacking neurons, with only a few cells observed close to the border (Fig. 1d-d'), contrasting with the uniform distribution of microglia (Fig. 1d"). The expression profile of 10 additional markers is summarized in Supplementary Table 1. Outside the perturbed regions, the distribution of neurons appeared normal, and the cell density was not significantly different from wild type (number of neurons per 0.01 mm 2 ; wild type, 39.2 ± 2.32; efnA2 KO, 38.01 ± 1.62, n = 3; Fig. 1e-e").
Ephrin-A2 modulates the transition of neocortical progenitors towards differentiation. The loss of neuronal cell labeling in the neocortex could indicate a developmental role for efnA2. We, therefore, examined the developmental expression of ephrin-A2 in the wildtype neocortex. At E14.5 ephrin-A2 was detected in the marginal zone (MZ) and in the VZ, comprised of apical progenitor cells ( Fig. 2a-a"). Intense ephrin-A2 expression persisted in the VZ at E16.5, with expression extending to the adjacent SVZ (Fig. 2b'). Ephrin-A2+ cells in the cortical plate (CP) also expressed the neuronal marker NeuN (Fig. 2b"), revealing that ephrin-A2 is present in both progenitor cells and differentiated neurons in the developing neocortex.
Failure of cells to exit the neurogenic compartment following ephrin-A2 knockdown suggests that ephrin-A2 signaling is necessary for newly generated neurons to migrate out of the neurogenic compartment. Alternatively, ephrin-A2 may act at an earlier stage of neurogenesis, with the reduction in ephrin-A2 resulting in progenitors failing to commit to a differentiated neuronal fate and arrest in the VZ.
To determine the identity of the sh_efnA2-GFP cells, we tested for the expression of progenitor markers and demonstrated that efnA2 knockdown cells persisting in the neurogenic zone maintain the expression of Pax6 (Fig. 2e-e"). In the embryonic neocortex, Pax6 expression is restricted to apical progenitors dividing at the ventricular surface 9 . Pax6 controls neocortical cell numbers by regulating the balance between proliferation and cell cycle exit 32 . Altogether, our results suggest that loss of ephrin-A2 causes progenitors to remain in the VZ and maintain Pax6 expression, which ultimately would result in increased proliferation at the expense of neuronal differentiation. Ephrin-A2 reverse signaling regulates neocortical progenitor proliferation. We further investigated the relationship between ephrin-A2 and cell cycle and whether this effect was mediated through forward or reverse signalling using a combination of in utero electroporation and in vitro approaches. To determine if ephrin-A2 knockdown at E15.5 affected the proliferation of apical progenitors, we quantified the proportion of proliferative cells within the GFP+ population in the VZ at 3dpe (Fig. 3a). The fraction of GFP+ cells expressing the proliferation marker Ki67 increased in sh_efnA2-GFP electroporated mice compared to controls (sh_ctrl-GFP, 0.37 ± 0.05; sh_efnA2, 0.53 ± 0.08, n = 5, p = 0.01, Mann-Whitney test; Fig. 3a'). This result confirms that reducing ephrin-A2 causes apical progenitors to remain in a proliferative undifferentiated progenitor state.
It is common for cells expressing ephrin ligands to co-express Eph receptors, segregated in separate microdomains to prevent cis-activation 33 . Therefore, the phenotype observed following loss of ephrin-A2 could result from a deficit in forward or reverse signaling. To discriminate between forward or reverse signaling in the control of apical progenitor differentiation, we dissociated the neocortices of E14.5 wild-type mice and plated the cells at low density to limit cell-cell interaction. Next, we added clustered recombinant frame chain (Fc) ephrin-A2 (ephrin-A2-Fc) to induce ephrin-A2 forward signaling (Fig. 3b'); reverse signaling by adding clustered EphA4-Fc ( Fig. 3b"); with clustered Fc fragment used as a negative control (Fig. 3b). While clustered EphA4-Fc has been demonstrated to activate ephrin-A2 reverse signaling 28 , it could potentially bind ephrin-A5 as well. However, the absence of ephrin-A5 expression in the embryonic neocortex between E13.5 and E16.5 34 ensured that ephrin-A2 reverse signaling was selectively activated in our culture system. BrdU was used to tag cells in the S-phase of the cell cycle. We found that the number of BrdU+ cells did not vary between control and ephrin-A2-Fc conditions. However, activation of ephrin-A2 reverse signaling significantly reduced the number of BrdU+ cells (control-Fc, 16.9 ± 0.94; ephrin-A2-Fc, 17.1 ± 1.3; EphA4-Fc, 11.3 ± 0.55, n = 3, p < 0.001, Kruskal-Wallis test; Fig. 3b"'). Therefore, the increase in proliferation of neocortical progenitors following ephrin-A2 knockdown is likely to be mediated via a reduction in ephrin-A2 reverse signaling rather than a reduction in forward signaling on Eph receptor bearing cells.
Despite cellular density appearing normal in the neocortices of ephrin-A2 knockdown animals (Fig. 4c), we detected discontinuities in the laminar distribution of GFP+ neurons in the electroporated hemispheres ( Fig. 4c'). The supergranular layers of ephrin-A2 knockdown neocortices exhibited discrete regions devoid of GFP + cells which were also devoid of NeuN + cells (Fig. 4c"), similar to the cortical defects (patches) we described in the neocortex of adult efnA2 KO (Fig. 1). Therefore, loss of ephrin-A2 in apical progenitors through targeted knockdown is sufficient to phenocopy the abnormal laminar architecture observed in the knockout animals.

Reduction of the calbindin-positive interneuron fraction in the supragranular layers of efnA2 KO mice.
To determine if loss of ephrin-A2 also affected interneurons, we compared calbindin immunolabeling between efnA2 KO and wild type control. We identified a severe reduction in interneurons density within the patches of reduced NeuN+ labeling in efnA2 KO ( Fig. 5a-a", hatched region). Calbindin + interneurons were present in the rest of the neocortex, exhibiting a distribution profile comparable to the wild type control with the majority of the cells populating the supragranular layers 2/3 and fewer cells in the infragranular layers 5/6 ( Fig. 5b-b'). However, the density of calbindin+ cells throughout the entire neocortex, excluding the abnormal patches, was significantly reduced (number of cells in layers 2 to 4 per 80,000 µm 2 ; wild type, 135.50 ± 4.96; efnA2 KO, 61.59 ± 14.17, p = 0.029, n = 4, Mann-Whitney test; Fig. 5b"). This suggests that ephrin-A2 plays a major role in the establishment of this fraction of inhibitory neurons but, unlike the focal effect observed in the case of excitatory neurons, loss of ephrin-A2 affects interneurons throughout the entire neocortex suggesting that the ligand has distinct roles in excitatory and inhibitory neurons development.
Ephrin-A2 is expressed by superficially migrating interneurons. At E14.5, interneurons emerging from two transient telencephalic regions, the medial ganglionic eminence (MGE) and the POA (Fig. 6a) and characterized by the expression of GAD 65 & 67 also express ephrin-A2 (Fig. 6a'; boxed region magnified in 6a"'). Ephrin-A2+ cells were organized in a continuous stream extending from the POA to dorsolateral telencephalon, (Fig. 6a"; open arrowheads), correlating with the superficial migratory stream. Interneurons migrating along the superficial migratory stream reach the neocortex at the level of the MZ as illustrated by the dense GAD65 & 67 staining observed in the MZ at E18.5 (Fig. 6b'). Double-labeling revealed the presence of ephrin-A2+ cells in the MZ at this stage, in addition to the dense population in the VZ and scattered throughout the CP observed at earlier stages (Fig. 6b"). Ephrin-A2 expressing cells in the MZ co-express GAD 65 & 67 (Fig. 6c-c") confirming their interneuronal identity. Altogether, this expression profile demonstrates that interneurons originating from the MGE and the POA upregulate ephrin-A2 expression after exiting the MGE and POA neurogenic zones, characterized by the high nuclei density (Fig. 6a) and the lack of GAD 65 & 67 expression (Fig. 6a'), as they join the superficial migratory stream to reach the neocortex. They maintain the ligand expression after completing the tangential phase of their migration to the MZ and dispersing radially in the CP. The lack of ephrin-A2 expression in the VZ of the 3rd ventricle suggests that the ligand does not participate in progenitor proliferation but might play a role in regulating tangential migration.
Ephrin-A2 promotes the migration of interneurons emerging from the POA. Interneurons emerging from the POA follow a superficial migratory stream along the ventral surface of the brain and are kept segregated from their MGE-born counterparts through EphA4-ephrinB3 mediated bidirectional repulsion 27 . To elucidate the function of ephrin-A2 in regulating the migration of POA-born interneurons we undertook an ex vivo electroporation approach, enabling us to specifically knockdown ephrin-A2 in the POA (Fig. 7a,b). After 2 days of in vitro culture (2div), we counted the number of GFP+ cells in 100µm-wide bins parallel to the medial edge of the VZ to quantify the extent of cell migration from the site of tissue electroporation (see schematic Fig. 7). Fewer than 50% of cells electroporated with control DNA remained within 100 µm of the medial edge of the VZ of the POA whereas more than 60% of cells electroporated with ephrin-A2 knockdown shRNA remained within this region (scrambled, 0.493 ± 0.054; sh-efnA2 0.629 ± 0.036, n = 15 sections/condition, p = 0.049, Student's t-test; Fig. 7c). The reduced migration of sh_efnA2 electroporated cells was also reflected by a significant decrease in the proportion of cells that had migrated from 200-300 µm (scrambled, 0.128 ± 0.018; sh-efnA2 0.059 ± 0.01, n = 15, p = 0.0034, Student t-test; Fig. 7c). Overall, our data suggest that knockdown of ephrin-A2 within interneurons emerging form the POA reduces their rate of tangential migration, rather than completely inhibiting their ability to reach the neocortex.

Discussion
Our study demonstrates that loss of ephrin-A2 during development leads to perturbations in the establishment of neocortical layers, resulting in the formation of discrete, random, patches depleted of neurons. Although Eph/ ephrins are traditionally known for regulating migratory events, our results suggest that alteration of the neocortical architecture in the absence of efnA2 results from the abnormal production of neurons before migration to the cortical plate is initiated. Although the lamination defects in efnA2 KO neocortex were spatially restricted to patches, with the neuronal density comparable to control animals in unaffected regions, we uncovered a significant loss of calbindin-expressing neocortical interneurons in apparently 'normal' regions of the neocortex, suggesting that in addition to differentiation of pyramidal neurons, ephrin-A2 also contributes to the establishment of neocortical interneurons.
Comparable local disruptions of the neocortical layers observed in efnA2; A3; A5 triple knockout mice 24 have been attributed to the abnormal lateral dispersion of excitatory neurons in the cortical plate, without specifying the role of each gene. The presence of ephrin-A2+ neurons in the cortical plate from E14.5 to E18.5, supports the hypothesis that the ligand participates in the regulation of neuronal migration. However, during these stages, ephrin-A2 is predominantly expressed in the VZ suggesting that it has additional functions. The VZ is comprised of Pax6+ apical progenitors 9 undergoing mainly asymmetric division (78%), to generate a self-renewed apical progenitor and either a neuron (65.8%) or a basal progenitor. Only 10% of divisions are symmetrical with both daughter cells remaining in the VZ 2,35 . Accumulation of proliferative Pax6+ cells in the VZ following reduction of efnA2 suggests that a larger proportion of apical progenitors divided according to a symmetrical self-renewing mode and re-entered the cell cycle instead of generating excitatory neurons.
Consistent with these findings, fewer neurons deriving from progenitors in the VZ were present in the postnatal neocortical layers, with dramatic disruption of the laminar architecture observed in extreme cases, replicating the efnA2 KO phenotype. Our results reveal that ephrin-A2 acts as an early cell-fate determinant in apical progenitors, directing their progeny towards a differentiated neuron identity and repressing cell division. The lack of ephrin-A2 signaling promotes cell proliferation and maintains the apical progenitor pool leading to abnormal neuron production and alteration of the neocortical architecture (see Fig. 8).
The signaling elicited by the interaction of ephrin-A2 with an Eph receptor is bidirectional, activating transduction pathways in both ligand and receptor-bearing cells. Although activation of EphA4 forward signaling by intrinsic ephrin-B1 30 or extrinsic ephrin-A5 34 promotes apical progenitor proliferation and neuron production in the mouse embryonic neocortex, the addition of clustered ephrin-A2 to dissociated embryonic neocortex cultures had no effect on cell proliferation. Consistent with evidence that ephrin-A2 reverse signaling inhibits the proliferation of adult stem cells in the mouse SVZ 36 , we observed reduced proliferation following activation of ephrin-A2, demonstrating that ephrin-A2 function on apical progenitors is mediated through reverse signaling. Ephrin-A2 is tethered to the cell membrane via a GPI anchor, preventing it from directly activating intracellular transduction molecules. Instead, activated ephrin-A2 molecules form complexes with other transmembrane molecules and "highjack" their signaling pathway 20 . The neurotrophin receptors p75 and TrkB, critical for neural progenitors commitment to a neuronal fate by inhibiting cell division and promoting neuronal maturation 37,38 , are privileged co-receptors for ephrin-A2 39,40 .
While ephrin-A2 reverse signaling is activated by EphA7 in the adult SVZ 36 , we were not able to identify a unique receptor responsible for eliciting this pathway in the embryonic neocortex with EphA3, A4, A5 and A7 all being strongly expressed in the neocortex. EphA3 is an interesting binding partner candidate for ephrin-A2 expressed on apical progenitors in the neocortical VZ. It has previously been demonstrated that ephrin-A2/ EphA3 interaction elicits the recruitment of the metalloproteinase ADAM10 resulting in cleavage of ephrin-A2 from the cell surface 41,42 . This molecular interaction could also underlie the observed scarcity of ephrin-A2 beyond the VZ in the mouse neocortex.
The significant reduction of calbindin labeling in efnA2 KO neocortex suggests that ephrin-A2 participates in the establishment of neocortical interneurons. Unlike our observations in the neocortex, ephrin-A2 is not expressed in the germinal layers of the regions where interneurons are born, principally the MGE 14,15 and the POA. Ephrin-A2 is detected in the superficially migrating interneurons, in agreement with recent demonstration that it promotes the motility of MGE-derived interneurons 28 . Our new evidence confirms that this role is conserved in POA-born interneurons. However, cell migration is not arrested in absence of ephrin-A2; it is only delayed with interneurons eventually reaching the neocortex, albeit later than normal. Therefore, the reduction in neocortical calbindin labeling is unlikely to correspond to an absence of interneurons due to abnormal migration.
Although the calbindin + population is reduced by 50% across the supergranular layers, the overall neuronal density is normal in efnA2 KO neocortex, beside the patches. Therefore, the decrease in calbindin + cells might not reflect an actual absence of supergranular interneurons but instead abnormal expression of the marker itself. As a member of the calcium-binding molecule family, Calbindin becomes upregulated in mature interneurons to buffer Ca 2+ flux 43 . Interneuron maturation is dependent upon correct synaptogenesis and pruning, which are both under the control of Eph/ephrin signaling 44,45 . In particular, ephrin-A2 reverse signaling utilizes the TrkB transduction pathway 40 known to modulates calbindin upregulation in maturing interneurons 46,47 . Similarly to efnA2 KO, TrkB deficient animals exhibit a reduction of calbindin + interneurons, consequence of the downregulation of the cell marker and not an actual loss of interneurons. In addition, delayed migration following loss of ephrin-A2 implies that interneurons are exposed to an aberrant, more mature, environment when they disperse through the neocortex. Therefore, the abnormal signals they are exposed to and the atypical network of connections they attempt to integrate are likely to affect the maturation of interneurons lacking ephrin-A2, ultimately affecting calbindin expression.
Thus, the dramatic interneuronal phenotype we report in efnA2 KO results from an impaired execution of maturation programs leading to lower than normal calbindin expression and not an actual depletion of neocortical interneurons.
efnA2 KO mice exhibit an interesting behavioral phenotype characterized by perturbed social interactions and repetitive grooming which reflects aspects of autistic-like behaviors in humans 48 . Members of the Eph/ephrin family, including EphA6 and EphB2, have been associated with Autism Spectrum Disorder (ASD) in recent genome-wide association studies (GWAS) 49,50 but no studies have yet linked ephrin-A2 with the disorder. It could be a consequence of technical limitations as sequences with high GC content (ephrin-A2 GC ratio = 67%) are poorly captured in exome sequencing studies 51 . The discrete and indiscriminate disruption of neuronal density we observed in efnA2 KO neocortex has, however, been reported in at least one systematic anatomical study of the neocortical architecture of children diagnosed with ASD 31 . The lamination defects they report in all ASD samples and absent from age-matched controls share many of the characteristics we observe in efnA2 KO mice, including the spatially restricted configuration with sharp borders and the stochastic distribution pattern. While the high degree of variability regarding neocortical areas and layers disrupted in ASD patients could be anticipated considering the large spectrum of symptoms observed in patients and the long list of genes associated with ASD, it is less expected in an animal model bearing a mutation in a single gene.
The phenotypic consistency observed between ASD samples and the efnA2 KO mice suggests a strong link between Eph/ephrin and ASD. Here, we demonstrate a novel mechanism by which ephrin-A2 regulates neurogenesis and determine that disruption of ephrin-A2 activity in the VZ is a potential contributor to ASD. Furthermore, our findings establish a new time-frame for the cellular mechanisms underpinning ASD, suggesting that they can occur as early as the initial stages of neocorticogenesis.

Materials and Methods
Mice. All experiments and procedures were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and were approved by Monash University and the University of Western Australia Animal Ethics Committees. Animals were housed in cages of 2-5 in a controlled environment with a 12 hour light/dark cycle. Pregnant mice were housed individually 48 h prior to surgery to allow habituation to their environment. Adult efnA2 homozygous knockout (background strain C57bl/6j) brains were obtained from the University of Western Australia. For in utero electroporation and tissue collection, time-mated 8 weeks old pregnant C57bl/6j mice were obtained from local colonies. The plug date was defined as E0.5, and the day of birth was defined as P0. A minimum sample size of n = 3 was used.
In utero electroporation. The enfA2 knockdown construct tagged with GFP was a gift from Professor Jürgen Bolz (see 28 for details). A GFP-tagged scrambled shRNA was used as a control. Mouse ephrin-A2 was amplified using the forward GGGCGGCAGATCTCCAAG and reverse AAAAGTCCACCCCACTCCC primers and the resulting 1564 bp fragment cloned into pCAGS-IRES-GFP. Pregnant mice were assigned a construct using a simple randomization method. Time-mated pregnant females were anesthetized with isoflurane (4% for induction and 2% to maintain) on a heated mat. The abdominal cavity was open and the uterine horns exposed. The plasmid suspension (0.5 µg.µL −1 in PBS) was injected in the lateral ventricle using a glass needle and an electric current (35 V, 50 ms, 950 ms interval ×5 pulses) was delivered using 1 mm platinum tweezertrodes (BTX) connected to EMX BTX (Harvard apparatus). Embryos were harvested 3 or 15 days post electroporation (dpe).
Ex vivo electroporation and slice assay. E14.5 embryonic brains were dissected in Complete HBSS medium (see 52 , the two hemispheres separated and the randomly assigned plasmid solution injected directly in the POA followed by application of an electric current (25 V, 50 ms, 250 ms interval ×4 pulses) using 1 mm platinum tweezertrodes (BTX). Electroporated hemispheres were embedded in 4% low-melting point agarose (LMP, Sigma) and sectioned using a vibratome into 300 µm thick coronal slices which were transferred on the membrane of a cell culture insert (Millipore) pre-coated with poly-L-lysine (100 µg.mL −1 ) and laminin (10 µg.mL −1 ). Slices were cultured for 48 hours in slice culture medium 52 at 35 °C and 5% CO 2 after which they were fixed with 4% PFA overnight at 4 °C, rinsed in PBS and mounted on microscope slides using Fluoromount-G mounting medium (SouthernBiotech).
Tissue preparation for immunolabeling. Animals received a lethal dose of sodium pentobarbital (100 mg.kg −1 ) and were transcardially perfused with 0.1 M heparinised sodium phosphate buffer (PBS) containing 0.1% sodium nitrite to optimise vasodilation, followed by 4% paraformaldehyde (PFA) in PBS. Mouse embryos harvested from time-mated females were anesthetized on, quickly decapitated, and the brains dissected. All brains were postfixed in 4% PFA for 24 h at 4 °C. Electroporated embryonic brains were embedded in 3% LMP and 100 µm sections cut using a Vibratome (Leica). Following post-fixation, wild type (non-electroporated) embryonic brains and all postnatal brains were cryoprotected in PBS-20% sucrose, embedded in optimal cutting temperature (OCT) medium, frozen in −45 °C isopentane and stored at −80 °C. Coronal cryostat sections of embryonic brains (18 µm) were collected on Superfrost slides and stored at −20 °C. Free floating sections (40 µm; coronal plane) were collected in serial order and stored at −20 °C in a cryoprotectant solution (50% 0.05 M phosphate buffer, 30% ethylene glycol, 20% glycerol).
Immunolabeling. Each staining was repeated at least 3 times independently. The representative images included represent results that were consistently observed. Free-floating sections were washed in PBS, while slide-mounted sections were rehydrated in PBS, before being incubated for 1 hour at room temperature with the blocking solution (10% Normal Goat Serum, 0.3% Triton X-100 in PBS). Due to low sensitivity, sections labeled with rabbit Anti-ALDH1L1 were incubated for 2 min in citrate buffer at 90 °C for antigen retrieval. The following primary antibodies were incubated overnight at 4 °C in the blocking solution: rabbit anti-ALDH1L1 In situ hybridisation. Digoxygenin-labelled riboprobe for murine efnA2 (NM_007909.3; nucleotides 158-1002) cloned into pGEMT was used as previously described 54 . efnA2 KO cell quantification. The experimenter was blinded to the genotype of the animals until after all the counting were completed. Low-magnification photomicrograph (1300 × 1030 dpi) of calbindin and NeuN stained sections of adult wild type and efnA2 −/− brains were acquired using a Zeiss Imager. Z1 microscope with an Axiocam HRm digital camera and Axiovision 4.8.2 software (Zeiss). Labeled cells were counted using the Fiji CellCounter plugin after applying a 10,000 µm 2 grid. 4 squares spanning the neocortical thickness were counted, 3 rows/images, 160 µm interval between sections, n = 3 for NeuN counting and n = 4 for calbindin. Statistical analysis and graphing of the data was performed using GraphPad Prism 6.0 for Mac OSX. The one-way non-parametric Mann-Whitney U test was used to compare two experimental conditions, for which the normal distribution could not be assumed.
In utero electroporation cell quantification. Coronal sections from electroporated brains collected at E18.5 (3 dpe) were incubated in Hoechst, mounted on glass microscope slides and coverslipped using Fluoromount mounting medium. Electroporated animals collected at P12 (15 dpe) were processed for immunolabeling, including GFP, as described above. For quantification of the number of GFP+ cells per compartment, low magnification images were captured to include the electroporated region from inner to outer surface, using a Zeiss Imager. Z1 microscope with an Axiocam HRm digital camera and Axiovision 4.8.2 software (Zeiss). Images were blinded using a script available in the free image analysis software Fiji. The entire electroporated region was counted, using a grid to facilitate the process. An average of 7.5 images/animal (between 5 and 10) was analyzed for E15.5+ 3 dpe experiments; 4 to 5 images were analyzed for E15.5+ 15 dpe. For the quantification of proliferative cells at E15.5+ 3 dpe, sections were imaged at higher magnification, Z-stacks were captured using an ApoTome and converted to maximum intensity projection. The resulting images were then processed as described above; with an average of 4 images/animal analyzed. The data were unblinded prior to statistical analysis and graphing using GraphPad Prism 6.0 for Mac OSX. The one-way non-parametric Mann-Whitney and Kruskal-Wallis tests were used to compare two experimental conditions and more than two experimental conditions, respectively. Slice assay cell quantification. Sections exhibiting extensive pyknosis, suggestive of cell death, revealed by Hoechst staining and sections where the electroporation was not restricted to the POA were systematically excluded from the analysis. Z-stacks of the electroporated regions were acquired with a Leica SP5 multichannel confocal microscope running the software LAS AF Lite. Fiji was used to generate maximum intensity projections. Using the freehand tracing tool in Illustrator (CS3), the outline of the electroporated region of the ventricular zone was drawn, defining the origin of migration (0 µm) and parallel lines were pasted at 100 µm interval. The images were saved in a tiff format. The number of GFP+ cells in each bin was counted using the CellCounter plugin in Fiji after applying a grid. The resulting counts were imported into Excel, unblinded and imported into GraphPad Prism 6.0 for Mac OSX for statistical analysis (unpaired Student's t-test) and graphing. Data availability. All relevant data are available from the authors.