Caroline Badouel

Christophe Audouard

Alice Davy
email: alice.davy@univ-tlse3.fr

Heterogeneity in the size of the apical surface of cortical progenitors

Keywords: ephrin, morphogenesis, mouse, neural progenitors

Background: The apical surface (AS) of epithelial cells is highly specialized; it is important for morphogenetic processes that are essential to shape organs and tissues and it plays a role in morphogen and growth factor signaling. Apical progenitors in the mammalian neocortex are pseudoepithelial cells whose apical surface lines the ventricle. Whether changes in their apical surface sizes are important for cortical morphogenesis and/or other aspects of neocortex development has not been thoroughly addressed.

Results: Here we show that apical progenitors are heterogeneous with respect to their apical surface area. In Efnb1 mutants, the size of the apical surface is modified and this correlates with discrete alterations of tissue organization without impacting apical progenitors proliferation.

Conclusions: Altogether, our data reveal heterogeneity in apical progenitors AS area in the developing neocortex and shows a role for Ephrin B1 in controlling AS size. Our study also indicates that changes in AS size do not have strong repercussion on apical progenitor behavior.

| INTRODUCTION

Epithelial cells or cells with epithelial features are widespread in metazoans. [START_REF] Tyler | Epithelium-the primary building block for metazoan Complexity1[END_REF] These cells are characterized by a polarized morphology with distinct basolateral and apical membranes and a tight cell-to-cell adhesion. The apical membrane or apical surface (AS) of epithelial cells is highly specialized, it harbors different organelles depending on the epithelial cell type, such as microvilli, a primary cilium or multiple cilia. Because numerous growth factor receptors, transporters, and channels are enriched apically in epithelial cells, the AS plays an important role in sensing environmental cues. In addition, the AS participates in organ morphogenesis during embryonic development. Indeed, it has been shown that constriction of the AS is a key step in epithelium folding and invagination. [START_REF] Martin | Pulsed contractions of an Actin-myosin network drive apical constriction[END_REF] While modifications in AS area have been tightly linked to morphogenesis of epithelial sheets in different contexts, [START_REF] Sawyer | Apical constriction: a cell shape change that can drive morphogenesis[END_REF] whether and how changes in AS area might also impact complex tissues has not been investigated thoroughly.

The developing neocortex, the dorsal part of the mammalian forebrain, is a complex tissue elaborated from a pseudo-epithelial sheet. Indeed, the neocortex is composed of a diversity of cells that originate from an initial pool of pseudo-epithelial neural progenitors also called apical progenitors. Apical progenitors are polarized, elongated cells, whose apical endfeet line the ventricular surface of the neocortex while their basal processes extend toward the pial surface. Apical progenitors either divide symmetrically, thereby amplifying their population, or asymmetrically to produce other cell types including intermediate progenitors and neurons. [START_REF] Miyata | Asymmetric of surface-dividing and non-surface-dividing cortical progenitor cells[END_REF][START_REF] Noctor | Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases[END_REF] As soon as they are generated, intermediate progenitors and neurons detach from the apical surface of the tissue and migrate basally, such that the apical (ventricular) surface of the tissue is formed exclusively by the tiling of apical progenitors AS. One special feature of the neuroepithelium is the existence of interkinetic nuclear migration (IKNM) whereby apical progenitors' nuclei migrate during the cell cycle from an apical location in mitosis to a basal location in S-phase. [START_REF] Sauer | Radioautographic study of Interkinetic nuclear migration in the neural tube[END_REF] Due to IKNM, the cytoarchitecture of apical progenitors AS changes over the cell cycle, broadening during mitosis and tightening during the other phases. [START_REF] Nagele | Ultrastructural changes in cells associated with interkinetic nuclear migration in the developing chick neuroepithelium[END_REF] As a result of proliferation and differentiation events, reorganization of the ventricular surface occurs constantly to accommodate apical mitosis and delamination events, impacting on tissue integrity and biophysical parameters, as shown for other epithelia. [START_REF] Pinheiro | Mechanical force-driven Adherens junction remodeling and epithelial dynamics[END_REF] In addition to these dynamic events, it has been shown that the mean size of AS area changes over the course of development in the mouse neocortex [START_REF] Nishizawa | Survey of the morphogenetic dynamics of the ventricular surface of the developing mouse neocortex[END_REF] and that it differs across brain regions, being smaller in the pallium than in the ganglionic eminence. [START_REF] Nagasaka | Comparison of the mechanical properties between the convex and concave inner/apical surfaces of the developing cerebrum[END_REF] Yet, the functional significance of these changes is not clear and very little is known on the mechanisms regulating the size of apical progenitors AS.

Ephrin B1 is a member of the Eph:Ephrin family which is involved in promoting cell adhesion or cell repulsion depending on the cellular context. [START_REF] Cayuso | Mechanisms of boundary formation by Eph receptor and ephrin signaling[END_REF][START_REF] Fagotto | Ephrin-Eph signaling in embryonic tissue separation[END_REF] In a previous set of experiments we reported that a fraction of Efnb1 mouse mutant embryos exhibit neural tube closure defects and microfolding of the ventricular surface, which we attributed to a loss of progenitor apical adhesion. [START_REF] Arvanitis | Ephrin-B1 maintains apical adhesion of neural progenitors[END_REF] Because ephrin B1 is enriched at the ventricular surface and Eph: Ephrin B1 signaling has been shown to promote actomyosin accumulation in different contexts, [START_REF] Cayuso | Actomyosin regulation by Eph receptor signaling couples boundary cell formation to border sharpness[END_REF][START_REF] O'neill | Unidirectional Eph/ephrin signaling creates a cortical actomyosin differential to drive cell segregation[END_REF] we hypothesized that it could control junctional tension between AP and potentially impact on AS size. Here we used en-face fixed and live imaging to quantitatively characterize the size of apical progenitors AS in wild type and Efnb1 mutant contexts. First, we show that apical progenitors in a wild type context are heterogeneous with respect to their AS areas and that this heterogeneity is not only linked to IKNM. We then use genetic tools to perturb Ephrin B1 expression and show that it modifies AS area without impacting on apical progenitors proliferation or differentiation. Moreover, mosaic loss of Ephrin B1 expression reveals strong non cell-autonomous feedback on AS size.

| RESULTS

| En-face analysis of cortical progenitors AS

To visualize the AS of apical progenitors, we exposed the ventricular surface by dissecting out the neocortex as illustrated in Figure 1A. Cortical explants were then stained with an F-actin probe, to delineate individual AS, and segmented to measure their sizes (Figure 1B). Quantification of apical areas revealed that the average apical domain is 4.4 μm 2 (±0.42 μm 2 ), with a median of 3.8 μm 2 at E13.5. Analysis of size distribution showed large variation in apical area sizes, with around 15% of very small AS (<2 μm 2 ) and around 20% of larger AS (>6 μm 2 ) (Figure 1C), indicating that at population level, AS area are heterogeneous. A previous systemic survey of AS area in the developing cortex indicated that apical surfaces tend to increase in size between E12 to E15. [START_REF] Nishizawa | Survey of the morphogenetic dynamics of the ventricular surface of the developing mouse neocortex[END_REF] Indeed, when we quantified AS areas 1 day later in development, at E14.5, we observed an increase in median AS area (Figure 1D). To identify the origin of this time-dependent variation in median AS area, we cultured embryonic cortical explants in vitro and performed live imaging with an en-face view of the ventricular wall (Figure 1E). We validated our culture conditions using an H2B reporter mouse line to visualize progenitor nuclei. We confirmed that apical progenitors divide properly, with intact interkinetic nuclear migration and division events (Movie S1 and data not shown). Development of apical domains were followed for 14 hours (Movie S2), and each image was segmented (Movie S3) to quantify epithelial growth dynamics. These movies revealed an increase in median AS area during the 14 hours time lapse (Figure 1D,E,F), indicating that the increase in AS area is intrinsic to the tissue. Comparison of the distributions of AS areas at the start of the movie (E14.5) and the end of the movie (E14.5 + 14H) revealed a shift in the proportion of larger AS area (from 14% of AS >8 μm 2 at E14.5 to 23% at E14.5 + 14H). These results are consistent with previously published data [START_REF] Nishizawa | Survey of the morphogenetic dynamics of the ventricular surface of the developing mouse neocortex[END_REF] indicating that even though we did not include live tissue explants in collagen, as done by other groups for en-face live imaging of apical progenitors, [START_REF] Nishizawa | Survey of the morphogenetic dynamics of the ventricular surface of the developing mouse neocortex[END_REF][START_REF] Konno | Neuroepithelial progenitors undergo LGN-dependent planar divisions to maintain self-renewability during mammalian neurogenesis[END_REF][START_REF] Fujita | Endfoot regeneration restricts radial glial state and prevents translocation into the outer subventricular zone in early mammalian brain development[END_REF] our experimental set up is valid. To further explore the dynamics of AS area over time, we tagged individual AS, chosen among those which did not go through cytokinesis during the time window, and followed their size (Movie S4 and Figure 2A,B; 50 AS analyzed). Automatic hierarchical clustering of the data using plotTwist revealed three subpopulations of AS: one subpopulation of AS did not change size over time, one subpopulation of AS showed a slight decreased size over time and one subpopulation of AS showed a slight increased size over time (Figure 2C). These data reveal that an increase in the proportion of larger AS and the increase in the size of a subpopulation of AS contribute to the increase in median AS area over time. Next, we wondered if heterogeneity in AS sizes could be linked to specific phases of the cell cycle or to mitosis. Apical progenitors' nuclei move to the ventricular surface to divide, which is accompanied by an enlargement of the apical surface. We measured the AS of cells before mitosis and their corresponding daughter cells after division for more than 20 dividing progenitors (Figure 2D,E and Movie S5). Sizes were plotted with T0 representing the time at which the AS of 2 daughter cells are first visible (Figure 2E). This revealed that, indeed, AS are larger prior to division and AS of daughter cells are on average half that size (Figure 2E). However, the persisting dispersion of AS areas throughout cell division (ranging from 5 to 20 μm 2 before division and from 1-12 μm 2 after division) suggests that heterogeneity in the size of AS area is not only the reflection of cell cycle progression and persists in interphase.

| Mutations in Efnb1 modifies AS sizes distributions

Because Eph: Ephrin B1 signaling has been shown to promote actomyosin accumulation in different contexts, we hypothesized that it could control junctional tension between AP and potentially impact on AS size. To investigate this hypothesis, we measured junctional tension using laser ablation in wild type and Efnb1 À/À mutant contexts (Figure 3A-C and Movies S6 and S7). Strikingly, recoil distance after ablation was on average twice longer in Efnb1 À/À mutants compared to wild-type controls (Figure 3B, initial recoil of 0.045 vs 0.22 in wildtype). When looking at the individual data at 60 seconds after the ablation, we noticed that the distance of recoil is not uniformly increased for all Efnb1 À/À ablated junctions. Instead, the range of recoiled distances is higher in Efnb1 À/À mutants compared to wildtype with a fraction of measurements that are higher than in wildtype whereas many recoil distances are similar in both genotypes (Figure 3C). To assess whether this difference in junctional tension underlies differences in AS size, we performed en-face fixed imaging on wild type, Efnb1 heterozygote and Efnb1 null contexts (Figure 3D) and analyzed AS of apical progenitors (Figure 3E). Quantification and analyses at population level revealed an increased median AS size (Figure 3F) and different distributions of AS area sizes (Figure 3F-G) in Efnb1 À/À and Efnb1 +/À embryos compared to wild-type control. Specifically, we observe a shift toward an increased proportion of large AS in ephrin B1 mutants. These results do not support the hypothesis that Ephrin B1 directly modulates apical constriction via the actomyosin network since increased junctional tension in this scenario should lead to increased constriction and smaller AS size in Efnb1 À/À embryos. Thus, this observation indicates that Ephrin B1 plays distinct roles in junctional tension and in the control of apical progenitors AS size.

| Mosaic ephrin B1 expression modifies AS area

To further assess the role of Ephrin B1 on the regulation of AS size, we focused on Efnb1 +/À heterozygote tissues which showed an unexpected distribution of AS sizes (Figure 3D-G). One particularity of Efnb1 +/À heterozygote tissues is that they are mosaic for Ephrin B1 expression. Indeed, Efnb1 is on the X chromosome and therefore subject to random X-inactivation. Moreover, Ephrin B1 positive and negative cells tend to sort out in development, creating patches of Ephrin B1 negative cells next to positive cells, [START_REF] Compagni | Control of skeletal patterning by ephrinB1-EphB interactions[END_REF][START_REF] Davy | EphrinB1 forward and reverse signaling are required during mouse development[END_REF] Figure 4A). Co-staining of Ephrin B1 and actin in the en-face view revealed a stereotypical spatial organization of patches of cells negative for Ephrin B1 (called B1-patches) surrounded by Ephrin B1 positive cells (Figure 4B). Strikingly, all B1-patches include clusters of cells with very small AS while cells at the periphery display larger AS (Figure 4B). Quantification at E13.5 showed a gradient in AS sizes from large in the first row outside the patch (B1+) to small at the center of the patch (B1À) (Figure 4C,E). At E13.5, AS have an average size of 2.6 μm 2 at the center of B1À patches, and 9.2 μm 2 in the first row of B1+ compared to 4.4 μm 2 in a wild-type cortex. This stereotypical organization is even more pronounced at E14.5, where the gradient ranges from 4 μm 2 at the patch center to 20 μm 2 at the periphery (compared to 5.4 μm 2 in average in wild-type, Figure 4D,E). Z-stacks analysis of B1À patches further revealed that more F-actin accumulates at the apical junctions of the cells at the center and F-actin extends more basally, compared to their neighbors (Figure 4F) consistent with increased apical constriction. Of note, the ventricular surface remains flat throughout the patches indicating that apical constriction is not the main driver of microfolding (Figure 4F).

It was unexpected to observe that only B1À cells at the center of the patch have a smaller AS but not B1À cells close to the periphery. We also observed that the AS of B1+ cells near the border were systematically larger than average. Taken together, these observations suggested that the modification in AS sizes could be nonautonomous. To test for this, we analyzed patches of B1+ cells surrounded by B1-territories (Figure 5 A). Quantification showed that these B1+ patches behave similarly to B1-patches, with a similar gradient in AS size at E13.5, from small at the center of the patch (3.7 μm 2 ) to large at the periphery (9.6 μm 2 ; Figure 5B,C). Together, these results suggest that quantitative differences in Ephrin B1 expression in neighboring cells lead to an increase in AS size, in both B1+ and B1À cells and that the decrease in AS size at the center of the patch is a non-cell-autonomous effect, secondary to the enlargement of AS at the periphery.

| Modification of AS area does not impact on proliferation or differentiation of apical progenitors

It has been reported previously that the mitotic index of cortical progenitors is decreased in Efnb1 mutants. [START_REF] Qiu | Regulation of neural progenitor cell state by ephrin-B[END_REF] To test whether modifications of AS size influence proliferation of apical progenitors, we performed P-H3 immunostaining on Efnb1 +/À explants which showed larger differences in AS size than Efnb1 À/À mutants. Mitotic F I G U R E 2 Heterogeneity in AS size is not due to cell cycle phases. A, En-face view of a E14.5 cortical slab cultured for 14 hours with an actin dye (SirActin). Images represent four different time frames from Movie S2. B, Zoom-in from A (white dashed region) with the skeleton superimposed (red). Tagged AS (green) were followed throughout the timelaps (Movie S4). C, Graphical representation of AS areas of 54 cells over the 14 hours movie. Lines in bold represent automatic hierarchical clustering of the 54 cells over time using plotTwist. D, Still images from Movie S5 with AS of a dividing apical progenitor highlighted in yellow. T0 corresponds to the time at which two AS were first observed after completion of cytokinesis. E, Measurement of AS area of 22 dividing apical progenitors before and after division. T0 corresponds to the time at which two AS were first observed after completion of cytokinesis figures could be observed at the center of B1À patches (Figure 6A) and B1+ patches (Figure 6B), suggesting higher apical constriction does not prevent progenitor divisions. Quantification of P-H3+ cells revealed that the mitotic index is similar inside and outside B1+ and B1À patches (Figure 6A,B), despite the fact that nuclei are located more basally inside the B1À patches (Figure 6C,D). In addition, immunostaining of coronal sections from Efnb1 +/À embryos with markers of apical (Pax6) and basal progenitors (Tbr2) showed no gross difference in B1+ and B1À patches (Figure 6E). Lastly, neuronal production, assessed with Ctip2 and Satb2 as markers of early born and late born neurons, respectively, appears similar in B1+ and B1À patches (Figure 6E). Altogether, these results show that modifications in AS size do not seem to correlate with changes in progenitor proliferation and/or differentiation.

| DISCUSSION

Differences in apical progenitors AS sizes over the course of development or in different brain region in the mouse have been reported previously. [START_REF] Nishizawa | Survey of the morphogenetic dynamics of the ventricular surface of the developing mouse neocortex[END_REF][START_REF] Nagasaka | Comparison of the mechanical properties between the convex and concave inner/apical surfaces of the developing cerebrum[END_REF] In addition, differences between species have been observed, for instance, AS tend to be larger in the mouse than in the ferret neocortex. [START_REF] Okamoto | Ferret-mouse differences in interkinetic nuclear migration and cellular densification in the neocortical ventricular zone[END_REF] However, none of these studies investigated what could be the mechanisms driving AS size changes.

Here we show that the AS of apical progenitors in the developing neocortex is heterogeneous in size ranging from 1 μm 2 to more than 20 μm 2 at mid-corticogenesis and that the median surface area increases over time, which is consistent with a previous study. [START_REF] Nishizawa | Survey of the morphogenetic dynamics of the ventricular surface of the developing mouse neocortex[END_REF] While some of this heterogeneity comes from the modification of AS during cell division, our live imaging data indicate that it is not the sole driver of AS size heterogeneity. One possible interpretation for apical progenitors with small and others with large AS areas, is that AS size reflects the existence of different apical progenitors. Indeed, several types of apical progenitors have been described in the neocortex based on morphological features. [START_REF] Xing | How neural stem cells contribute to neocortex development[END_REF] For instance radial glial cells extend a basal process all the way to the pial surface of the neocortex while apical intermediate progenitors or subapical progenitors harbor a shorter basal process. [START_REF] Gal | Molecular and morphological heterogeneity of neural precursors in the mouse neocortical proliferative zones[END_REF][START_REF] Pilz | Amplification of progenitors in the mammalian telencephalon includes a new radial glial cell type[END_REF] The absence of specific molecular markers for each type of apical progenitor precludes a definitive conclusion, yet, it has recently been shown in the primate neocortex that the transition from neuroepithelial cells to apical progenitors is accompanied by a decrease in AS area, [START_REF] Benito-Kwiecinski | An early cell shape transition drives evolutionary expansion of the human forebrain[END_REF] supporting the notion that different types of neural progenitors have different morphological features including different AS sizes.

Regardless of the type of apical progenitors, how might AS size be regulated? Here, we identified ephrin B1 as a molecular player acting on apical progenitor AS size. In Efnb1 homozygous null embryos, the distribution of AS areas is shifted, compared to the wild type context, with a higher proportion of apical progenitors with large AS area. This is consistent with observations made in Xenopus laevis embryos showing that loss of ephrin B2 prevents apical constriction and leads to neural tube closure defects. [START_REF] Ji | EphrinB2 affects apical constriction in Xenopus embryos and is regulated by ADAM10 and flotillin-1[END_REF] Further, it was shown previously in sea urchin embryos that Eph:ephrin signaling promotes apical constriction of ciliary band cells in an actomyosindependent manner, [START_REF] Krupke | Eph-Ephrin signaling and focal adhesion kinase regulate actomyosin-dependent apical constriction of ciliary band cells[END_REF] revealing a potential mechanism by which ephrin B1 could control apical progenitors AS size in the mouse. However, when assessing junctional tension in Efnb1 À/À tissue as a proxi for cortical actomyosin contraction, we observed an increased tension, which is not consistent with a relaxation of actomyosin-driven apical constriction. Another possible explanation for the increased AS size in EfnB1 À/À explants could be that absence of ephrin B1 increases cell adhesion. It is known that adhering cells tend to spread onto a surface or

F I G U R E 3
The distribution of apical progenitors AS size is modified in Efnb1 mutants. A, Laser ablation experiments on apical surfaces of cortical explants at E14.5 (Movies S6 and S7). Laser pulses (white arrows) were applied in the mid-point between two polygonal apices and junctional actin was visualized using SirActin. Scale bar = 5 μm. B, The distance between the two separated apices (recoil) was measured for 80 seconds after laser ablation. Means ± SEM; fitted non linear curve is shown. Average initial recoil is higher in Efnb1 À/À (0.045-95% CI 0.035-0.059) than in Efnb1 +/+ (0.022-95% CI 0.018-0.027) tissues. C, 60 seconds post ablation, average recoil is higher in Efnb1 À/À than in Efnb1 +/+ tissues. Each dot represents one ablated junction. n = 45 in Efnb1 À/À tissue and n = 62 in Efnb1 +/+ tissue, three independent experiments for each genotype. Unpaired t test, P < .0001. D, En-face view of cortical explants from E13.5 Efnb1 +/+ , Efnb1 +/À and Efnb1 À/À embryos stained for actin. Scale bar = 10 μm. E, Color-coded representations of AS areas in the three genotypes (different images than in D), calculated after segmentation, from light blue (0 μm 2 ) to dark red (23 μm 2 ). Scale bar = 10 μm. F, Violin plot of AS area (Log10) in the three genotypes with mean in red. Wilcoxon rank sum test with continuity correction, two-sided test to compare median was performed on non transformed data. P values are indicated on the graph. G, Cumulative distributions of AS sizes in the three genotypes (from at least four different embryos per genotype at E13.5; 2-3 images for each cortex, 167 096 cells total). Asymptotic two-sample Kolmogorov-Smirnov test. P values are indicated on the graph increase their surface of contact with their as was shown for E-cadherin-mediated adhesion. [START_REF] De Vries | Maternal β-catenin and E-cadherin in mouse development[END_REF] Since Eph: Ephrin B1 signaling induces the shedding of Ecadherin via ADAM10, [START_REF] Solanas | Cleavage of Ecadherin by ADAM10 mediates epithelial cell sorting downstream of EphB signalling[END_REF] absence of Ephrin B1 may increase cadherin -mediated adhesion of apical progenitors AS.

Our data in Efnb1 +/À tissue support this hypothesis. Indeed, in this mosaic situation, we observe typical differential adhesion-based sorting out of Ephrin B1 positive and Ephrin B1 negative AS, this correlates with changes in AS size. However, unexpectedly, both B1+ and B1-cells exhibit enlarged AS at patches boundary, which suggests that modification of adhesion at the B1+/ B1À boundary has non-autonomous consequences on neighboring cells. One striking non-autonomous consequence is apical constriction of AS in the center of the patches, independently of the Ephrin B1 expression status within constricted cells. A similar phenotype has been reported previously for Echinoid clones in Drosophila wing disc epithelium. It was shown that Echinoid +/À and Echinoid À/À cells at clonal boundary had enlarged apical surfaces while Echinoid À/À cells inside the clone had reduced apical surfaces. [START_REF] Wei | Echinoid is a component of adherens junctions that cooperates with DE-cadherin to mediate cell adhesion[END_REF] In addition, when ed clones grew large, apical constriction disappeared. Given that Echinoid is a component of adherens juncton that cooperate with DE-cadherin in cell adhesion, these observations further suggest that ephrin B1 non autonomously modulates AS size via a role on cell adhesion.

In addition to intrinsic molecular control, mechanical cues exerted at the ventricular surface of the tissue may also contribute to the modification of apical progenitors AS area. These mechanical cues include pressure from the cerebrospinal fluid as well as physical constraints exerted laterally and/or radially as the tissue grows through division and differentiation of neural progenitors. [START_REF] Abuwarda | Mechanobiology of neural development[END_REF] For instance it was recently proposed that IKNM fluidizes the neuroepithelium at early stages of development, when proliferation rates are high, while at later stages when proliferation rates decline, the tissue effectively solidifies. [START_REF] Bocanegra-Moreno | Cell cycle dynamics controls fluidity of the developing mouse neuroepithelium[END_REF] One possible role for Ephrin B1 in apical progenitors may be to provide buffering to mechanical perturbations and contribute to maintain AS area within a certain range. At tissue level, the severe apical constriction at the center of the B1+ and B1-patches leads to an altered positioning of apical progenitors nuclei and to microfolding of the ventricular surface. [START_REF] Arvanitis | Ephrin-B1 maintains apical adhesion of neural progenitors[END_REF] As a consequence, nuclei divide at more basal positions in B1+ and B1À patches, yet this does not change proliferation or differentiation rate, unlike what was shown in the zebrafish. [START_REF] Hiscock | Feedback between tissue packing and neurogenesis in the zebrafish neural tube[END_REF] Altogether, our data reveal heterogeneity in apical progenitors AS area in the developing neocortex and shows a role for Ephrin B1 in controlling AS size. Our study also reveals that changes in AS size in Ephrin B1 mutants does not seem to have strong repercussion on apical progenitor behavior.

| EXPERIMENTAL PROCEDURES

| Animals

Wild-type (Efnb1 +/+ ), heterozygote female (Efnb1 +/À ), homozygote female (Efnb1 À/À ), and hemizygote male (Efnb1 Y/À ) were described previously [START_REF] Davy | EphrinB1 forward and reverse signaling are required during mouse development[END_REF] and kept in a mixed 129S4/C57BL/6J genetic background. For clarity in embryonic studies, Efnb1 À/À refers to Efnb1-null embryos of both genders. Mice were housed in the CBI animal facility. Animal procedures were approved by the appropriate Ethics committee (APAFIS#1289-2 015 110 609 133 558 v5) and carried out in accordance with the European Communities Council Directive of November 24, 1986 (86/609/EEC). None of the procedures used in this study caused pain.

| Cortical explant dissection and staining

Timed-pregnant Efnb1 +/À mice, crossed to Efnb1 y/À males, were sacrificed in a CO 2 euthanasia chamber. Embryos at embryonic day 13.5 and 14.5 were surgically removed and placed in ice-cold PBS. The embryonic brain was dissected and fixed for at least 2 hours in 4% PFA. Further dissection of the brain was performed to obtain cortical explants. Explants were then fixed in 2% PFA for an additional 2 minutes and permeabilized in PBST (PBS + 0.1% Triton X100) for 10 minutes. Following a blocking step in PBST +5% CSF, explants were incubated with primary antibodies in the blocking buffer for 2-3 days. After 40 minutes washes, secondary antibodies were added in blocking buffer for 1 hour at room temperature. Then, explants were gently mounted under coverslips in Mowiol, and dried over-night at 4 before imaging on the Zeiss 710 Big confocal microscope, with a Â63 objective.

| Antibodies

Goat α-Ephrin B1 (R&D Systems AF-473, 1/50); Rabbit α-Phospho-Histone 3 (Cell Signaling 9701, 1/200).

| Explant culture

Timed-pregnant wild-type mice were sacrificed in a CO 2 euthanasia chamber. Embryos at embryonic day 14.5 were surgically removed and placed in ice-cold PBS. Cortical explants were dissected and incubated in DMEM:F2 media, supplemented with a cocktail of Penicillin/ streptomycin and 100 nM of SirActin in a 37 C incubator with 5% CO 2 . After 3 hours incubation, explants were placed apical side down on optimized live imaging polymer-coated dishes (from Ibidi) and maintained under meshed gold baskets for live imaging.

| Live Imaging

For time-lapse, explants were incubated in SiR-actin (SiR F-Actin labelling 50 nmol SiR-actin Kit, Sirochrome, SC001) according to the manufacturer's recommendations. Images were acquired on an inverted microscope (Leica inverted DMI8) equipped with a heating chamber (set up at 37 C, 5% CO 2 , and RH 90%), a spinning disk confocal head (CSU-X1-M1N, Yokogawa) a SCMOS camera and a 63X oil immersion Plan-Apochromat objective (NA 1.4-0.7). We recorded 15 μm thick z stacks (0.5 μm zsteps) at 5 minutes intervals (for 20 hours), with a pixel size of 120 nm (63X).

F I G U R E 6 Modification of AS size does not impact proliferation or differentiation of apical progenitors. A, En-face view of Efnb1 +/À cortex at the level of mitotic nuclei. Cortical slabs were stained for DNA (blue), actin (grey), Phospho-Histone H3 (P-H3, red), and Ephrin B1 (green). Scale bar = 10 μm. Percentage of P-H3+ nuclei in Ephrin B1 negative and Ephrin B1 positive territories. Quantification was done on 2684 B1À and 5115 B1+ cells from at least 3 different clusters. Data are showing averages ± SD. ns: not statistically significant. Student ttest. B, En-face view of Efnb1 +/À cortex at the level of mitotic nuclei. Cortical slabs were stained for DNA (blue), actin (grey), Phospho-Histone H3 (P-H3, red) and Ephrin B1 (green). Scale bar = 10 μm. Percentage of P-H3+ nuclei for Ephrin B1 positive cell inside and outside patches. Quantification was done on 6263 B1+ cells inside patches and 5409 B1À cells outside patches from at least 15 different clusters. Data are showing averages ± SD. ns: not statistically significant. Student t test. C, Apical view of Efnb1 +/À cortex, stained for actin (grey) and Ephrin B1 (green), Z reconstructions are shown below the X/Y images, at the cut indicated by a red dashed line. Scale bar = 10 μm. D, Quantification of the distance between nuclei and the apical surface in B1+ and B1À territories. Each dot is a nucleus. E, Coronal sections of E14.5 Efnb1 +/À cortex stained for Pax6, Tbr2, Ctip2 or Satb2, as indicated. Nuclei are stained with DAPI. Scale bar = 100 μm

| Photo-ablation

For photo-ablation, explants were treated as for liveimaging except that they were placed on small dishes with a glass bottom. Laser ablation experiments were performed using a pulsed Lined Q switch Yag double laser (532 nm, pulse length 0.4 ns, repetition rate up to 7 kHz, 7 μJ/pulse) steered by a galvanometer-based laser scanning device (Ilas2, Roper Scientific), mounted on a Leica DMI6000B inversed microscope. The laser beam was focused through an oil-immersion lens of high numerical aperture (Plan-Apochromat Â100/0.7-1.4 Imm Oil, from Leica).

Photo-ablation of apical junctions was done in the focal plane following a line of 1.6 μm for 40 ms at 30% laser power (total of 10 iterations, with a thickness of 1). Live images were acquired on the wide-field microscope with a CCD HQ2 cooled Ropper cameras with a pixel size of 64.5 nm/pixel, using a Cy5 filter and HBO illumination, limiting photobleaching. Acquisition was performed every 1 second during 2 seconds, before ablation, and, every second during 10 seconds, then every 10 second during 70 seconds, after ablation. Data analysis was performed according to the protocol described previously [START_REF] Liang | Measurement of mechanical tension at cell-cell junctions using two-photon laser ablation[END_REF] with the ImageJ software using MtrackJ Plugin.

| Image analysis

Huygens, IMARIS, and ImageJ software were used for image processing and data analysis.

For AS size measurement, we extracted from the 15 μm z-stack 2-3 actin stained images at the most apical z-levels, with minimal pollution from more basal actin network. Averaged actin images were then segmented using Tissue Analyzer plugin in ImageJ. [START_REF] Aigouy | Segmentation and quantitative analysis of epithelial tissues[END_REF] Global apical areas and AS of tagged cells were calculated automatically from segmented images using the EpiTools plugin in Icy. [START_REF] Heller | EpiTools: an open-source image analysis toolkit for quantifying epithelial growth dynamics[END_REF] Dividing apical surfaces were selected based on the following criteria: constriction of round apical surfaces followed by appearance of two smaller apical surfaces and correlating with progression through mitosis at nuclei level. AS size measurement of dividing cell was done by manually tracing cell limits in ImageJ. Segmentation of images of B1+ and B1À patches by rank was done manually. Ephrin B1 immunostaining and actin immunostaining were overlaid and AS at the boundary were segmented first, thus creating rank three of the bar plots.

| Statistical analyses

Non normality of the distributions of AS areas was tested using the Shapiro-Wilk normality test. Distributions were compared with Asymptotic two-sample Kolmogorov-Smirnov test for the cumulative plot and with Wilcoxon rank sum test with continuity correction, two-sided to compare the median for the violin plots. Automatic hierarchical clustering with euclidian distance was performed using plotTwist. [START_REF] Goedhart | PlotTwist: a web app for plotting and annotating continuous data[END_REF] Statistical analyses, data management, and graphical representations were done using R, Excel or GraphPad. Statistical tests are indicated in the legend of each Figure.

F

  I G U R E 1 AS of apical progenitors is heterogeneous in size. A, Schematic representation of the dissection procedure to visualize the AS of apical progenitors in an "en-face" view. B, En-face view of an E13.5 cortical slab stained for actin (left) and color-coded from light blue (0 μm 2 ) to dark red (23 μm 2 ), as calculated after segmentation (right). Scale bar = 10 μm. C, Distribution of AS areas, measured from five different embryos at E13.5 (2-3 images for each cortex, >200 000 cells total). Error bars indicate SEM. Average 4.4 μm 2 ± 0.21 À Median 3.8 μm 2 . D, AS sizes at E13.5 compared to E14.5 and E14.5 + 14 hours. Violin plots with mean in red. Wilcoxon rank sum test with continuity correction, two. sided test to compare median. P values are indicated on the graph. E, Apical view of a E14.5 cortical slab stained for actin, before (T0) and after 14 hours in culture (E14.5 + 14 hours). Scale bar = 10 μm. F, Distribution of AS sizes, averages from three different E14.5 cortical explants in culture (three images for each, >3000 cells total). Data represent average ± SEM

F I G U R E 4

 4 Modification of AS size in EfnB1 +/À mosaic contexts. A, Coronal section of E13.5 Efnb1 +/À brain stained for Ephrin B1 (green) and DNA (blue), Scale bar = 500 μm. B, Apical view of Efnb1 +/À cortex, at E13.5 (left) and E14.5 (right), stained for actin (grey) and Ephrin B1 (green). A drawing of some cells was superimposed on the actin staining (bottom), with a color code to show the different layers of cells at the boundary of Ephrin B1À patches (B1À cells in red and B1+ cells in green). Scale bar = 20 μm. C, D, Average sizes of AS relative to their rank from the cluster boundary at E13.5 (C) and E14.5 (D). Measurements were done for three clusters at E13.5 and 6 at E14.5, data are showing averages ± SEM. Sydak's multiple comparisons test, adjusted P-values ****P < .0001; ***P < .001; **P < .01. E, Schematic representation of cell layers at the boundary of the ephrin B1-patches with the layers numbered as in C and D. F, Apical view of Efnb1 +/À cortex, stained for actin (grey) and Ephrin B1 (green), Z reconstructions are shown below the X/Y images, at the level indicated by the white dashed line. Scale bar = 20 μm

F I G U R E 5

 5 The modification of AS size in EfnB1 +/À mosaic contexts is not cell autonomous. A, Apical view of Efnb1 +/À cortex at E13.5 stained for actin (grey) and Ephrin B1 (green). A drawing of some cells was superimposed on the actin staining (right), with a color code to show the different layers of cells at the boundary of the Ephrin B1+ patch (B1+ cells in red and B1-cells in green). Scale bar = 10 μm. B, Average sizes of AS relative to their rank from the cluster boundary. Measurement was done for 3 clusters, and data are showing averages ± SEM. Sydak's multiple comparisons test, adjusted P-values ****P < .0001; *P < .05. C, Schematic representation of cell layers at the boundary of Ephrin B1 positive clusters with the layers numbered as in B

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ACKNOWLEDGMENTS

The authors would like to thank members of the Davy team, especially Thomas Jungas for informal discussions and for insightful comments on the manuscript and Fraha Haidar for technical help. Thomas Mangeat and the Microscopy Core Facility LITC (Light Imaging Toulouse CBI) of CBI (Centre de Biologie Intégrative de Toulouse) provided invaluable help with laser ablation experiments. We thank the CBI-bigA facility engineers for their help and feedback with statistical analyses and data visualization. The authors would also like to thank the Anexplo-CBI animal facility platform for housing the mice. This work was funded by the Fondation pour la Recherche Médicale (DEQ20180339174) and by core funding from CNRS and Université Paul Sabatier.

AUTHOR CONTRIBUTIONS

Caroline Badouel: Conceptualization (equal); data curation (equal); formal analysis (equal); methodology (lead); writingoriginal draft (supporting); writingreview and editing (equal). Christophe Audouard: Data curation (equal); formal analysis (equal); investigation (supporting); methodology (supporting). Alice Davy: Conceptualization (equal); data curation (equal); formal analysis (equal); funding acquisition (equal); project administration (equal); resources (equal); supervision (equal); validation (equal); writingoriginal draft (equal); writingreview and editing (equal).

FUNDING INFORMATION

This work was funded by the Fondation pour la Recherche Médicale (DEQ20180339174) and by core funding from CNRS and Université Paul Sabatier.

CONFLICT OF INTEREST

The authors declare no conflict of interests.

DATA AVAILABILITY STATEMENT

Data are available on request to the corresponding author.

ETHICAL STATEMENT

Animal procedures were approved by the appropriate Ethics committee (APAFIS#1289-2 015110609133558 v5) and carried out in accordance with the European Communities Council Directive of November 24, 1986 (86/609/EEC).

ORCID

Alice Davy https://orcid.org/0000-0003-2134-4526

SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.