Abstract

Using a fetal brain slice culture system that recapitulates early cortical plate (CP) development, we screened the “Diversity Set” chemical library from the National Cancer Institute in order to identify molecules that interfere with radial migration and CP formation and identified 11 candidate molecules. Although most compounds had broadly similar effects, histological and immunohistochemical studies with preplate and neuronal differentiation markers disclosed some differences in the anomalies induced, suggesting that the identified molecules may act on different targets. Selected compounds were tested for activity on signaling pathways known to be important during radial migration and CP development, namely reelin, phosphatidylinositol 3-kinase/Akt-protein kinase B(PKB)/glycogen synthase kinase-3ß (GSK3β), atypical protein kinases C (aPKC), and Cdk5. No perturbation of reelin signaling or GSK3β activity was detected. One molecule decreased the phosphorylation of Akt and focal adhesion kinase and may act via direct or indirect inhibition of Cdk5, whereas another inhibited phosphorylation of aPKCζ/λ and may interfere with cell polarity and leading edge formation or progression. These molecules potentially provide new tools to study a neuronal migration and CP development.

Introduction

The development of the cerebral cortex follows a complex sequence. Neuronal precursors proliferate in ventricular and subventricular zones located around the lateral ventricles. Postmitotic neurons leave the germinal zones and reach their destination by following complex routes (Caviness and Rakic 1978; Caviness 1982; Lambert de Rouvroit and Goffinet 2001; Nadarajah and Parnavelas 2002; Rakic 2003). The majority of cortical neurons migrate along radial fibers and settle, first, in the preplate (PP), where they contribute to a population of reelin-negative pioneer cells, and then in the cortical plate (CP), where they differentiate into glutamatergic neurons. Other cells, such as Cajal Retzius cells, are generated in the cortical hem and around the hilus of the hemispheric vesicles and migrate tangentially in a subpial location in the PP and cortical marginal zone (MZ) (Grove and Fukuchi-Shimogori 2003; Meyer and others 2004; Bielle and others 2005). γ-aminobutyric acidergic interneurons, generated in the ganglionic eminences, move in 2 stages, first tangentially at the border of the hemispheric subventricular zone and then radially toward the CP (Anderson and others 1997; Nadarajah and Parnavelas 2002). Like other motile cells, neurons move by using at least 2 interdependent cellular mechanisms (Lambert de Rouvroit and Goffinet 2001). Migration begins with the projection of a leading edge, a highly sophisticated process that is based on recognition of microenvironmental cues and integration of these cues to regulate the actin treadmill (Guan and Rao 2003; Govek and others 2005). Migration itself occurs when the nucleus engages into the leading edge. This nuclear movement, often referred to as “nucleokinesis” (Morris and others 1998; Bellion and others 2003; Tsai and Gleeson 2005), is accompanied by a movement of the microtubule organizing center (MTOC) (Xie and Tsai 2004). Whereas nucleokinesis is thought to rely on microtubule dynamics, recent work shows that it is inhibited by blebbistatin, implicating myosin II in the process (Bellion and others 2005). At the end of radial migration, neurons settle in the PP and CP, the organization of which requires normal reelin signaling (Curran and D'Arcangelo 1998; D'Arcangelo 2001; Tissir and Goffinet 2003).

Increasing numbers of small molecular weight inhibitors are becoming available to probe a wide variety of signaling pathways. Although they lack the exquisite specificity of antibodies, or of plasmids encoding interfering RNA, dominant negative or positive proteins, small inhibitors have advantages: they often diffuse freely in cells where they act rapidly; they are relatively easy to use in vitro and in vivo; and they provide a starting point for pharmacological developments. Using a slice culture system that recapitulates several features of early cortical development, small inhibitors were used previously to demonstrate the role of atypical protein kinases C (aPKC) and Src family kinases in neuronal migration and reelin signaling (Jossin and others 2003a). Here, we used that in vitro system to screen a chemical library and identified 11 original leading compounds that interfere with radial neuronal migration and/or early CP development. Such molecules should prove useful to define the signaling partners implicated in these developmental events.

Materials and Methods

Animals

Animal procedures were carried out in accordance with institutional guidelines and ratified by competent animal ethics committees. Mice were outbred CD1, maintained on a normal irradiated diet with unrestricted access to water. Previous studies showed that embryonic development proceeds somewhat more rapidly in CD1 than in inbred mouse strains and that an immature CP is already present in the lateral telencephalic wall at E13.5. In order to obtain embryonic brains at the PP stage, prior to any significant migration to the CP, a CD1 colony was maintained on an inverted light cycle (light “off” from 7 AM to 5 PM), and females were inspected for the presence of vaginal plugs between 5 and 7 PM. Embryos at 13 days of pregnancy (E13) were used for slice preparation.

The Chemical Library

We used the Diversity Set developed by the Developmental Therapeutics branch of the National Cancer Institute (NCI). This set is composed of 1992 leading compounds selected from approximately 140 000 compounds of the NCI drug depository. Details on the selection, structures, and activities of the molecules included in the Diversity Set can be found on the NCI Developmental Therapeutics Program website (http://dtp.nci.nih.gov). Stock molecules are provided in 96-well microplates, at a concentration of 10 mM in pure dimethyl sulfoxide (DMSO).

Screening Using Embryonic Brain Slice Culture

The slice culture system was described previously (Jossin and others 2003a). Briefly, brains from E13 embryos were rapidly dissected out on ice, embedded in 4% low-melting agarose (Promega, Leiden, The Netherlands) prepared in Dulbecco's modified Eagle's medium: F12 (DMEM-F12) (with glutamine, glucose and hepes, Cambrex, Eupen, Belgium), and glued on a vibratome support. Sections (of 300-μm thickness) were cut in the coronal plane and laid on collagen-coated polytetrafluoroethylene membranes (Transwell-COL, Costar 3494). The culture medium was DMEM-F12 supplemented with B27 (1/50), G5 (1/100), penicillin, and streptomycin (all from Cambrex or Invitrogen, Carlsbad, CA). Culture in the presence of different concentrations of DMSO showed that this solvent does not interfere with slice development up to a concentration of 1% (v/v). The screening was conducted at a target concentration of 10 μM, allowing to test pools of 8 compounds (at a final DMSO concentration of 0.8%) added to 2 slices per well. During screening of the last 6 plates (containing the last 461 molecules), it appeared that too many pools were toxic. This part of the screening was pursued by excluding from the screen organometallic molecules and by decreasing the test concentration to 2 μM. Cultures were carried out for 2 days in vitro (DIV) in a chamber (MIC-101, Billups-Rothenberg, Del Mar, CA) continuously gassed with water-saturated 95%O2–5%CO2, that was itself placed in a cell culture incubator. For each positive pool, active molecules were identified by testing separately the 8 (or 5) components at 10 μM (or 2 μM foas indicated above), and the positive molecules were then assayed at different concentrations from 1 to 50 μM. In all experiments, controls included a slice processed without culture to check developmental stage and quality of preparation and 2 slices cultured in medium plus DMSO alone at 0.8–1% concentration.

Activity In Vivo

The molecules selected from the screen were administered intraperitoneally to pregnant females on 3 consecutive days, at stages E12, 13, and 14. Each injection aimed at achieving a concentration comparable with that tested in vitro, assuming free diffusion of the compounds in body water. E15 fetuses were examined by macroscopic inspection and by histology as described below.

Histology and Immunohistochemistry

Samples were fixed in Bouin's fluid for 2 h prior to embedding in paraffin. 8 -thick Serial sections (of 8-μm thickness) were collected on 4 slides. One slide was stained with hematoxylin-eosin (HE), and the others were used for immunohistochemistry. The following antibodies were used: mouse monoclonal antichondroitin sulfate (chondroitin sulfate proteoglycan [CSPG], clone CS-56, Sigma 8035), mouse monoclonal antibody against neuronal class III ß-tubulin (Tuj1, COVANCE mms-435P), mouse monoclonal anti–microtubule-associated protein 2 (Map2, clone HM-2, Sigma M4403), polyclonal rabbit anti-Tbr1 (generous gift of Dr R. Hevner), and mouse monoclonal anti-bromodeoxyuridine (BrdU) (Becton Dickinson, Mountain View, CA). For BrdU-labeling experiments, BrdU was administered to pregnant females at the dose of 40 μg/g body weight, 2 h prior to sacrifice. For immunohistochemistry, slides were deparaffinized, incubated with 3% H2O2 for 30 min, blocked for 30 min in 5% normal goat serum in phosphate buffered saline (PBS, pH 7.4), and incubated with primary antibodies overnight. Detection was carried out with an avidin-biotin-peroxidase kit (Vectastain ABC, Vector Laboratories, Burlingame, CA), using diaminobenzidine as the chromogen.

Preparation of Slice Lysates, Western Blot, and Immunoprecipitation

E13 brain slices were cultured, as described above, for 1 DIV and then lyzed for 10 min at 4 °C in nonidet-p40 (NP40) buffer composed of 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% NP40, 0.08% Na3VO4, 0.1% NaF, 1 mM phenylarsine oxide, 25 mM NaPPi, 80 mM ß-Glycerol phosphate, 0.1 μM okadaic acid, and 2 mM proteinase inihibitor with ethylenediamine tetraacetic acid (Complete, Roche, Vilvoorde, Belgium). Lysates were clarified by centrifugation at 14 000 g for 15 min at 4 °C, and protein concentration was measured by the Bradford method. Samples corresponding to 30 μg proteins were analyzed on 8% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane (BioScience) by electroblotting (Invitrogen). Membranes were blocked with 5% low-fat milk and 0.01% Tween20, in PBS, for 30 min, and incubated overnight at 4 °C with antibodies. After washing, secondary horseradish peroxidase-conjugated antibodies (DakoCytomation, Heverlce, Belgium.) were applied for 35 min, and membranes were washed, treated with the SuperSignal West Pico chemiluminescent substrate (Pierce), and exposed to Hyperfilm enhanced chemoluminescence (Amersham Biosciences). Reelin signaling was studied by estimating tyrosine phosphorylation of the Dab1 adapter (Howell and others 1999, 2000), and other pathways were assessed using the following antibodies: anti-Akt (Santa Cruz sc-1618) and phospho-Akt (Ser473) (Cell Signalling Technology 9271); anti–glycogen synthase kinase-3ß (GSK3ß) and phospho-GSK3ß (Ser9) (Cell Signalling Technology 9332 and 9336); anti-Tau and phospho-Tau (Ser396) (Biosource 44752G and AHB0042); anti-PKCζ and phospho-nPKCζ (Thr 410), that cross-react with PKCλ/ι (Santa Cruz sc216G and sc12894R); and anti–focal adhesion kinase (FAK) (Santa Cruz sc558) and phospho-FAK (Ser732) (Biosource 44-590G). For the Dab1 phosphorylation assay, 35 μg total protein were incubated with a rabbit polyclonal antibody raised against a C-terminal peptide of Dab1, overnight at 4 °C, followed by an incubation with protein A–agarose beads (Roche) for 2 h. The beads were washed three times with NP-40 buffer. Proteins were eluted by boiling for 2 min in PAGE loading buffer and analyzed on 8% SDS-PAGE. The proteins were transferred to nitrocellulose membrane, and Dab1 was detected with a mouse monoclonal anti-Dab1 antibody (E1) or with an antiphosphotyrosine monoclonal antibody (4G10, UBI). All experiments were carried out at least in triplicate. Autoradiography films were scanned, and signals were quantified using the Scion National Institutes of Health (NIH) program. In each experiment, signal of phosphorylated and total protein was normalized to that obtained in the control situation; data were expressed as the ratio of phosphorylated versus total protein and analyzed using Student's t-test.

Results

The Control Situation: Cortical Development In Vitro

Previous work showed that cortical development in slices recapitulates several features of normal development in vivo, such as radial CP organization, PP splitting, and inside–outside maturation (Jossin and others 2003a). In the present screening, slice development was assessed histologically using HE staining. In addition, staining with anti-CSPG antibodies was used to visualize PP splitting (Sheppard and Pearlman 1997), and neuronal maturation was estimated by immunostaining with the Tuj1 antibody, which labels early postmitotic neurons (Lee and others 1990) and is expressed in migrating neurons (Moody and others 1989). Map2 staining was used to disclose early dendritic maturation and to identify tangentially migrating cells from the ganglionic eminences (Tamamaki and others 1997). At E13, the CP had not yet developed, and the cortex was composed of the ventricle zone (VZ) and the PP (Fig. 1A1); CSPG immunoreactivity (Ir) was seen in the whole cerebral wall but was clearly stronger in the PP than in the VZ (Fig. 1B1). Both Tuj1- and Map2-Ir cells were present in the PP. The VZ contained no Tuj1-Ir cells and sparse Map2-Ir cells that formed a layer in the VZ as described (Tamamaki and others 1997) (Fig. 1C1, D1). After 2 DIV, a dense CP populated with radial neurons was developed in the external field of telencephalon, bracketed between an external MZ and an inner subplate (SP) followed by the intermediate zone (IZ) of migration and the VZ (Fig. 1A2). CSPG labeling was weak in the MZ, absent in the CP, and maximal in the SP; the signal decreased progressively through the IZ, and the VZ was almost negative (Fig. 1B2). Tuj1-Ir and Map2-Ir were both the most prominent in the MZ and decreased progressively in the CP, SP, and upper IZ; the lower tiers of the IZ contained labeled Tuj1 and Map2 positive, bipolar cells with tangentially directed processes (Tamamaki and others 1997), and the VZ was negative (Fig. 1D2). The gradient of CP maturation was estimated by labeling the neurons generated just before slice preparation with BrdU and by staining adjacent sections with antibodies against Tbr1, that labels early postmigratory neurons, and against anti-BrdU. At the start of the culture, Tbr1-Ir cells were present in the PP, and BrdU-labeled cells were restricted to the upper part of VZ. In normal slices after 2 days of culture (Fig. 2B,B′), BrdU-positive cells that reached the CP migrated to the external tiers of the CP and were positioned above strongly Tbr1-Ir cells, reflecting the inside–outside maturation gradient (Nowakowski and others 1975; Caviness and Rakic 1978; Caviness 1982).

Figure 1

Effect of the selected molecules, C1–C11, on neuronal migration in slices. (A1–A13) HE staining to appreciate overall morphology; (B1–B13) Immunostaining with anti-CSPG antibody to assess PP splitting; (C1–C13) Immunostaining for Tuj1 to disclose neuronal differentiation; and (D1–D14) Anti-Map2 staining to show early dendritic maturation and tangential migration from ganglionic eminence. (A1–D1) Show control embryonic brain slices at E13 (0 DIV); (A2–D2) Show control slices cultured for 2 DIV with DMSO only. After 2 DIV (A2), a well developed CP appeared, and PP was split into MZ and CP (B2); postmitotic neurons were located in MZ, CP, SP, and upper IZ (C2); some tangentially migrating bipolar neurons appeared in upper IZ (D2). Compounds C1–C11 perturbed CP formation in vitro (A3–A13), resulted in defective PP splitting (B3–B13); and induced variable anomalies in Tuj1 and Map2 labeling, with occasional ectopic neurons in inner IZ and VZ (C3–C13, D3–D13). Bar = 100 μm.

Figure 1

Effect of the selected molecules, C1–C11, on neuronal migration in slices. (A1–A13) HE staining to appreciate overall morphology; (B1–B13) Immunostaining with anti-CSPG antibody to assess PP splitting; (C1–C13) Immunostaining for Tuj1 to disclose neuronal differentiation; and (D1–D14) Anti-Map2 staining to show early dendritic maturation and tangential migration from ganglionic eminence. (A1–D1) Show control embryonic brain slices at E13 (0 DIV); (A2–D2) Show control slices cultured for 2 DIV with DMSO only. After 2 DIV (A2), a well developed CP appeared, and PP was split into MZ and CP (B2); postmitotic neurons were located in MZ, CP, SP, and upper IZ (C2); some tangentially migrating bipolar neurons appeared in upper IZ (D2). Compounds C1–C11 perturbed CP formation in vitro (A3–A13), resulted in defective PP splitting (B3–B13); and induced variable anomalies in Tuj1 and Map2 labeling, with occasional ectopic neurons in inner IZ and VZ (C3–C13, D3–D13). Bar = 100 μm.

Figure 2

Effects of C1–C11 on the cortical maturation gradient. Pregnant mice were injected with BrdU at E13, 2 h prior to slice preparation (BrdU(−2 h)). At the time of slice preparation, PP cells are Tbr1 positive (A) and BrdU-positive cells, presumably in S-phase are located in the outer VZ (A′). After 2 days in vitro (2 DIV), in control slices incubated with DMSO, both PP and CP cells are Tbr1 positive (B), and several BrdU-positive neurons have migrated to the outer part of the CP, beyond older and highly Tbr1-positive cells (B′), reflecting inside–outside maturation. In slices incubated in the presence of C1–C11 (panels CM), early neuronal differentiation is relatively preserved as indicated by Tbr1 staining. BrdU-positive cells are found in the IZ and in the inner part of the CP, but very rarely reach the outer level of the CP, indicating that migration is disturbed and that CP maturation proceeds from outside to inside. Bar = 100 μm

Figure 2

Effects of C1–C11 on the cortical maturation gradient. Pregnant mice were injected with BrdU at E13, 2 h prior to slice preparation (BrdU(−2 h)). At the time of slice preparation, PP cells are Tbr1 positive (A) and BrdU-positive cells, presumably in S-phase are located in the outer VZ (A′). After 2 days in vitro (2 DIV), in control slices incubated with DMSO, both PP and CP cells are Tbr1 positive (B), and several BrdU-positive neurons have migrated to the outer part of the CP, beyond older and highly Tbr1-positive cells (B′), reflecting inside–outside maturation. In slices incubated in the presence of C1–C11 (panels CM), early neuronal differentiation is relatively preserved as indicated by Tbr1 staining. BrdU-positive cells are found in the IZ and in the inner part of the CP, but very rarely reach the outer level of the CP, indicating that migration is disturbed and that CP maturation proceeds from outside to inside. Bar = 100 μm

Identification of Molecules That Affect Cortical Development In Vitro

Following the screening procedure described in Materials and Methods, we identified 77 compounds that were toxic to brain slices when tested individually at a concentration of 2–10 μM. Forty-seven of them were known to be toxic from the literature. These toxic molecules were not considered further. We also identified 11 molecules that perturbed migration and/or the formation of the CP and were selected for further study. A summary with references in the Developmental Therapeutics Program library (NSC number), most reproducible active concentrations in the slice culture assay, chemical names, and formulas, is provided in Table 1. In order to facilitate description, the 11 candidate molecules will be referred to as compounds C1–C11. As shown in Figure 1A3–A13, in the presence of compounds C1–C11, the development of slices proceeded relatively normally, in that the thickness of the telencephalic wall and the overall cellular morphology were comparable with those of normal slices. BrdU-labeling experiments confirmed comparable tracer incorporation of labeled cells in normal slices and in the presence of selected compounds (Fig. 2). In contrast, architectonic development was drastically affected. The most evident anomaly was the poor definition of the CP, that was populated with obliquely oriented neurons, and traversed by aberrant fiber bundles, a phenotype reminiscent of that observed in reeler embryos (Caviness 1976; Lambert de Rouvroit and Goffinet 1998) and in slices treated with PP2, a Src family blocker, or with PKC inhibitors (Jossin and others 2003b). All compounds resulted in some degree of defective PP splitting, as estimated by CSPG staining, although some minor differences between compounds were observed (Fig. 1B3–B13). Molecules C5, C6, C10, and C11 resulted in an abnormal expression of CSPG-Ir in the VZ, where no CSPG-Ir cells were detected in normal slices. In slices incubated with C7, very little CSPG staining was found outside the MZ. Some but not all molecules perturbed the pattern of neuronal maturation estimated with Tuj1 and anti-Map2 staining. In the presence of C1–C11, Tuj1-Ir was prominent in the MZ, CP, SP, and upper IZ, like in control slices (Fig. 1C3–C13). However, in the presence of compounds C2–C8 and C10, intensively Tuj1-Ir neurons were also detected in the deep IZ and the VZ. The results of Map2 immunostaining (Fig. 1D1–D13) were quite similar to those obtained with Tuj1. The presence of Tuj1 and Map2-Ir cells in the lower IZ and VZ presumably reflected ectopic differentiation of postmitotic neurons that failed to migrate past the IZ or to leave the VZ, a feature that is not found in reelin-deficient brains or slices. In most experiments, the orientation of slices did not consistently allow visualization of tangentially migrating, Map2-positive cells from ganglionic eminences, and the effect of C1–C11 on that migratory stream could not be studied. The effects of C1–C11 on the gradient of CP maturation were studied using BrdU-labeling experiments carried out as explained in Materials and Methods. In slices treated with C1–C11, early postmigratory cells stained normally with Tbr1 antibodies, confirming that early neuronal differentiation was relatively unaffected. However, BrdU-positive cells migrated consistently in the intermediate zone or to the inner tiers of the CP, but did not reach its outer part, indicating inverted, outside–inside maturation (Fig. 2).

Table 1

The 11 identified molecules that perturb neuron migration in vitro

graphic 
graphic 

Compounds C1–C11 Do Not Perturb Cortical Development In Vivo

To assess whether molecules C1–C11, active in vitro, also affected development in vivo, they were administered intraperitoneally to pregnancy-dated females at E11, 12, and 13, and fetuses were examined at E15. No overt malformation was identified by macroscopic inspection, and no pathological anomaly was found in their brains, which appeared histologically indistinguishable from those of normal E15 controls.

Effects of C1–C11 on Signaling Pathways Known to Be Implicated in Cortical Development

As a first attempt to identify putative targets of C1–C11, we studied their effect on 4 pathways that are known to play critical roles during early cortical development, namely, the reelin pathway, Akt/protein kinase B–GSK3β signaling, aPKCs, and Cdk5 signaling. Analyses were done at least in triplicate, and modifications were considered significant only when they were detected in all experiments (Fig. 3). Reelin signaling, or at least its proximal component estimated by comparing tyrosine phosphorylation of the Dab1 adapter in control situation and in the presence of C1–C11, was unaffected. In some cases, a decrease of Dab1 phosphorylation was observed, but it occurred always in parallel with a decrease of Dab1 protein levels, whereas inhibition of reelin signaling results in upregulation of Dab1 protein levels (Howell and others 2000; Jossin and others 2003b). Reelin activates phosphatidylinositol 3-kinase signaling, resulting in phosphorylation and activation of the kinase Akt (also named protein kinase B), which, in turn, inhibits GSK3ß by phosphorylation on serine 9 (Beffert and others 2002; Bock and others 2003). The effects of C1–C11 on this pathway were tested by examining phosphorylation of Akt on serine 473, GSK3ß on serine 9, and the microtubule associated protein Tau on serine 396, a site known to be phosphorylated by GSK3ß (Ishiguro and others 1992; Takahashi and others 1995, 2000). With the notable exception of C4, which decreased the phosphorylation of Akt, the selected compounds had no significant effect on these signaling molecules, confirming that they did not perturb known components of reelin signaling. PKCs of the atypical family are involved in rearrangement of the cytoskeleton, in neuronal polarity (Etienne-Manneville and Hall 2001, 2003; Manabe and others 2002; Shi and others 2003; Suzuki and others 2003), and in radial neuronal migration (Jossin and others 2003b). Therefore, the influence of compounds C1–C11 on phosphorylation of aPKCζ/λ/ι was tested, and compound C10 consistently resulted in a decreased phosphorylation signal, with upregulation of protein content (Fig. 2). Another reproducible effect was obtained by incubation with compound C4, which consistently resulted in decreased phosphorylation of FAK at Ser372, a site phosphorylated by Cdk5, an enzyme important for the formation of the CP (Fig. 2) (Ohshima and others 1996; Chae and others 1997).

Figure 3

Effect of C1–C11 on selected signaling pathways. At E13, embryonic brain slices were cultured with the 11 selected compounds for 1 DIV, and lysates were analyzed for phosphorylation of putative target proteins implicated in reelin signaling or neuronal migration. Data are plotted as the ratio of phosphorylated over total protein and normalized to the control situation. Incubation with C4 decreased phosphorylation of Akt and FAK and culture in the presence of C10 inhibited phosphorylation of aPKCs.

Figure 3

Effect of C1–C11 on selected signaling pathways. At E13, embryonic brain slices were cultured with the 11 selected compounds for 1 DIV, and lysates were analyzed for phosphorylation of putative target proteins implicated in reelin signaling or neuronal migration. Data are plotted as the ratio of phosphorylated over total protein and normalized to the control situation. Incubation with C4 decreased phosphorylation of Akt and FAK and culture in the presence of C10 inhibited phosphorylation of aPKCs.

Discussion

Neuronal migration and positioning requires the coordinated action of multiple cellular proteins and signaling pathways, such as the reelin- and Cdk5-dependent cascades (Rice and Curran 2001; Gupta and others 2002; Ohshima and Mikoshiba 2002; Gupta and Tsai 2003; Tissir and Goffinet 2003; Xie and Tsai 2004). Large numbers of low–molecular weight molecules are continuously developed, and some prove very useful to probe signaling in different settings. Here, we used an in vitro mouse embryonic brain slice culture (Jossin and others 2003a) to screen a chemical library, in order to identify molecules that interfere with early cortical development, aiming to obtain leading compounds from which series of analogs could be developed. The chemical bank selected is the Diversity Set from the Developmental Therapeutics Program of the National Cancer Institute (NIH, USA). This set of 1992 compounds was selected from the ∼140 000 compounds of the NCI repository, using chemical prediction programs. The screen, mostly conducted at a target concentration of 10 μM, identified several molecules with potent toxicity for embryonic brain tissue, that were not considered for further analysis. It is worth noting that this series included molecules such as camptothecin, bouvardin, cucurbitacin, ellipticin, topotecan, the quinocarmycin analog DX-52-1, and the mitomycin derivative T53, known to block cell proliferation in cancer models. The screening resulted in the selection of 11 molecules, named C1–C11, that perturb deeply radial neuronal migration and/or CP formation and have not been described previously. Very little chemical data are available on them (Table 1) and not informative in terms of putative mechanisms of action. The fact that the chemical structures are very different may suggest that they act on different biochemical targets.

As a first attempt to define better the action of compounds C1–C11, we performed histological studies using HE stain and well-validated antibodies that reflect neuronal differentiation and maturation and allow an estimation of the radial gradient of CP maturation. Apart from some differences noted below, compounds C1–C11 had largely similar effects. At the active dose, none of them appears to affect dramatically cell death or proliferation because a comparable development of telencephalic tissue and similar BrdU incorporation occurred in slices cultured with or without them. All 11 compounds inhibited PP splitting to various extends and often dramatically. Defective PP splitting is sometimes considered pathognomonic of defective reelin signaling, and is not found, for example, in mice with defective Cdk5 signaling. Because C1-C11 had no consistent effect on Dab1 phosphorylation, other pathways besides reelin or downstream of Dab1 may be implicated in PP splitting. Another common effect of C1–C11 was to perturb the architectonic organization of the developing CP and to result in a maturation that proceeds from outside to inside, whereas normal cortical maturation proceeds from inside to outside. Despite these common morphological features on PP splitting and CP formation, there were some differences in the malformations induced by C1–C11, particularly in the numbers of ectopic, prematurely differentiated neurons in the intermediate and ventricular zones. These defects were not observed in vivo following intraperitoneal injections in pregnant mice. Several reasons may explain this absence of effect, such as rapid degradation or difficulty to cross the placenta. Preparation of more diffusible and/or stable analogs is needed for further in vivo studies.

Morphological differences in vitro may reflect different mechanisms of action of C1–C11, as indicated also by the preliminary biochemical analysis. By probing key-signaling molecules with known roles in cortical neuronal migration, we identified 1 target affected by compound C10, namely, phosphorylation of aPKC in the activation loop and 2 targets consistently affected by compound C4, namely, phosphorylation of FAK at Ser372 and of Akt at Ser473. The inhibition of phosphorylation of aPKC by C10 might reflect inhibition of PDK1 or other kinases capable of phosphorylating this site (Balendran and others 2000). aPKC inhibition perturbs migration in slices in culture, in which it generates a reeler-like phenotype (Jossin and others 2003b). However, compound C10 did not inhibit Dab1 phosphorylation and resulted in ectopic neuronal maturation in the VZ, indicating that it perturbs migration in a reelin-independent manner, or downstream of Dab1. Observations in other systems have shown that aPKC is required for translocation of the MTOC in the direction of and possibly prior to the extension of a leading edge (Etienne-Manneville and Hall 2003; Henrique and Schweisguth 2003; Solecki and others 2004; Suzuki and others 2004), and this mechanism is likely to be important in radial migration. However, the phosphorylation of aPKC has not been studied in this context. Phosphorylation of FAK at Ser372 is a recognized target of the kinase Cdk5, which, together with its coactivators p35 and p37, plays a key role in neuronal migration (Ohshima and others 1996; Chae and others 1997; Ko and others 2001). Phosphorylation of FAK by Cdk5 is important for microtubule organization, nuclear movement, and neuronal migration (Xie and others 2003; Nikolic 2004; Xie and Tsai 2004). To our knowledge, no specific inhibitors of Cdk5 signaling are currently available because inhibitors such as Roscovitin also block other Cdk-related enzymes. Like that of FAK(Ser372), the inhibition of Akt(Ser473) phosphorylation by C4 was consistent and significant. This could reflect a decrease in Akt activity. However C4 does not decrease GSK3β phosphorylation at Ser9, indicating that the inhibition of Akt is partial. High concentrations of C4 were toxic and did not allow us to test this further. The observation that C4 prevents PP splitting, a phenotypic trait that is not present in Cdk5 mutant mice, indicates that it also affects other unrecognized signaling components.

In sum, in the present work, we identified 11 original compounds that interfere with cortical development in vitro. Future work should focus on the development of analogs with increased activity and on the identification of the biochemical target of these molecules.

We thank the Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, USA, particularly R. Schultz and J. Johnson, for gift of the chemical library and selected compounds and for advice, and Robert Hevner for gift of the anti-Tbr1 antibody. We also thank Esther Paître for technical assistance and members of the Developmental Neurobiology laboratory for discussion. YJ is Postdoctoral Researcher at the Fonds National de la Recherche Scientifique. This work was supported by grants Fonds de la Recherche Fondamentale Collective 2.4504.01, Action de Recherches Concertées 02/07-276, and by the Fondation Médicale Reine Elisabeth, all from Belgium. Conflict of Interest: None declared.

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