Identification of Small Molecules ThatInterfere with Radial Neuronal Migrationand Early Cortical Plate Development

Libing Zhou, Yves Jossin and Andre M. Goffinet

Developmental Neurobiology Unit, Louvain University Medical

School, Avenue East Mounier, 73, Box DENE7382, B1200

Brussels, Belgium

Using a fetal brain slice culture system that recapitulates earlycortical plate (CP) development, we screened the ‘‘Diversity Set’’chemical library from the National Cancer Institute in order toidentify molecules that interfere with radial migration and CPformation and identified 11 candidate molecules. Although mostcompounds had broadly similar effects, histological and immuno-histochemical studies with preplate and neuronal differentiationmarkers disclosed some differences in the anomalies induced,suggesting that the identified molecules may act on differenttargets. Selected compounds were tested for activity on signalingpathways known to be important during radial migration and CPdevelopment, namely reelin, phosphatidylinositol 3-kinase/Akt-protein kinaseB(PKB)/glycogen synthase kinase-3ß (GSK3b), atypicalprotein kinases C (aPKC), and Cdk5. No perturbation of reelinsignaling or GSK3b activity was detected. One molecule decreasedthe phosphorylation of Akt and focal adhesion kinase and may act viadirect or indirect inhibition of Cdk5, whereas another inhibitedphosphorylation of aPKCz /l and may interfere with cell polarityand leading edge formation or progression. These moleculespotentially provide new tools to study a neuronal migration and CPdevelopment.

Keywords: atypical PKC, Cdk5, chemical library, Dab1, reelin,slice culture

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 cor-

tical marginal zone (MZ) (Grove and Fukuchi-Shimogori 2003;

Meyer and others 2004; Bielle and others 2005). c-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 path-

ways. 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 de-

velopment. Such molecules should prove useful to define the

signaling partners implicated in these developmental events.

Materials and Methods

AnimalsAnimal 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 LibraryWe used the Diversity Set developed by the Developmental Therapeu-

tics branch of the National Cancer Institute (NCI). This set is composed

Cerebral Cortex January 2007;17:211--220

doi:10.1093/cercor/bhj139

Advance Access publication February 15, 2006

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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 CultureThe 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-lmthickness) 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 de-

velopment up to a concentration of 1% (v/v). The screening was

conducted at a target concentration of 10 lM, 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 lM. 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 lM (or 2 lM foas

indicated above), and the positive molecules were then assayed at

different concentrations from 1 to 50 lM. 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 VivoThe molecules selected from the screen were administered intraperi-

toneally 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 ImmunohistochemistrySamples were fixed in Bouin’s fluid for 2 h prior to embedding in

paraffin. 8 -thick Serial sections (of 8-lm 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 mono-

clonal 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 ex-

periments, BrdU was administered to pregnant females at the dose of

40 lg/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, andImmunoprecipitationE13 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

mMTris--HCl pH 7.5, 150 mMNaCl, 1% NP40, 0.08% Na3VO4, 0.1% NaF, 1

mM phenylarsine oxide, 25 mMNaPPi, 80 mM ß-Glycerol phosphate, 0.1

lM 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 corre-

sponding to 30 lg 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 en-

hanced 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-PKCf and phospho-nPKCf(Thr 410), that cross-react with PKCk/i (Santa Cruz sc216G and

sc12894R); and anti--focal adhesion kinase (FAK) (Santa Cruz sc558)

and phospho-FAK (Ser732) (Biosource 44-590G). For the Dab1 phos-

phorylation assay, 35 lg total protein were incubated with a rabbit

polyclonal antibody raised against a C-terminal peptide of Dab1, over-

night 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 nitrocel-

lulose 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 ex-

pressed 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,

212 Small Inhibitors of Cortical Development d Zhou and others

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 maturationand 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 tangentiallymigrating 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 variableanomalies in Tuj1 and Map2 labeling, with occasional ectopic neurons in inner IZ and VZ (C3--C13, D3--D13). Bar = 100 lm.

Cerebral Cortex January 2007, V 17 N 1 213

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 pro-

gressively 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,B9), 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

Figure 1. Continued.

214 Small Inhibitors of Cortical Development d Zhou and others

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 slicepreparation, PP cells are Tbr1 positive (A) and BrdU-positive cells, presumably in S-phase are located in the outer VZ (A9). After 2 days in vitro (2 DIV), in control slices incubatedwith 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 (B9),reflecting inside--outside maturation. In slices incubated in the presence of C1--C11 (panels C--M), 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 maturationproceeds from outside to inside. Bar = 100 lm.

Cerebral Cortex January 2007, V 17 N 1 215

gradient (Nowakowski and others 1975; Caviness and Rakic

1978; Caviness 1982).

Identification of Molecules That Affect CorticalDevelopment 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 lM.

Forty-seven of themwere 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 con-

centrations in the slice culture assay, chemical names, and for-

mulas, 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 ob-

served 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. How-

ever, BrdU-positive cells migrated consistently in the interme-

diate zone or to the inner tiers of the CP, but did not reach

its outer part, indicating inverted, outside--inside maturation

(Fig. 2).

Compounds C1--C11 Do Not Perturb CorticalDevelopment In Vivo

To assess whether molecules C1--C11, active in vitro, also

affected development in vivo, they were administered intraper-

itoneally 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 histologi-

cally indistinguishable from those of normal E15 controls.

Effects of C1--C11 on Signaling Pathways Known to BeImplicated 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--GSK3b 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 upregu-

lation 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 mole-

cules, 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 aPKCf/k/i was tested, and compound C10 consistently resulted in

a decreased phosphorylation signal, with upregulation of pro-

tein 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 phos-

phorylated by Cdk5, an enzyme important for the formation of

the CP (Fig. 2) (Ohshima and others 1996; Chae and others

1997).

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

216 Small Inhibitors of Cortical Development d Zhou and others

Table 1The 11 identified molecules that perturb neuron migration in vitro

NSC number Chemical name Formula

C1 48300 4,49-Methylenebis (phenylarsonic acid)

C2 329070 (3Z)-3-hydrazono-3H-indol-2-yl phenylcarbamate

C3 19832 [1-cyclohexyl-3-(2-nitrophenyl)aziridin-2-yl](phenyl)methanone

C4 339585 2-f[3-(dimethylamino)propyl]aminog-1,4-dihydroxyanthra-9,10-quinone

C5 65537 3-[(E)-(4-amino-3-methoxy-1-naphthyl)diazenyl]benzenesulfonamide

C6 47118 N9-1-naphthyl-N,N-diphenylcarbamimidic chloride

C7 116709 2-(1-benzoyl-1,2-dihydroquinolin-2-yl)-1-phenylethanone

C8 56452 9-isobutyl-6-f[(2-methyl-1-naphthyl)methyl]thiog-9H-purin-2-amine

C9 136476 2-((3-(2-(dimethylamino)benzyl-2-(pyridin-4-yl)-tetrahydropyrimidin-1(2H)-yl)methyl)-N,N-dimethylbenzeneamine

C10 93355 17-(2-Aminothiazol-4-yl)-11-hydroxy-10,13-dimethyl-1,7,8,10,11,12,13,15,16,17-decahydro-2H-cyclopenta[a]phenanthren-3(6H,9H,14H)-one, salt with 4-bromobenzensulfonic acid

C11 172033 4,49,4$,49$-(ethane-1,1,2,2-tetrayl)tetrakis(2,6-dichlorophenol)

Cerebral Cortex January 2007, V 17 N 1 217

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 con-

ducted at a target concentration of 10 lM, 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 avail-

able 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 bio-

chemical 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, com-

pounds C1--C11 had largely similar effects. At the active dose,

none of them appears to affect dramatically cell death or pro-

liferation 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 down-

stream 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 forma-

tion, 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 defectswere 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, phosphoryla-

tion 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 migra-

tion in slices in culture, in which it generates a reeler-like

phenotype (Jossin and others 2003b). However, compound C10

Figure 3. Effect of C1--C11 on selected signaling pathways. At E13, embryonic brainslices were cultured with the 11 selected compounds for 1 DIV, and lysates wereanalyzed for phosphorylation of putative target proteins implicated in reelin signaling orneuronal migration. Data are plotted as the ratio of phosphorylated over total proteinand normalized to the control situation. Incubation with C4 decreased phosphorylationof Akt and FAK and culture in the presence of C10 inhibited phosphorylation of aPKCs.

218 Small Inhibitors of Cortical Development d Zhou and others

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 cur-

rently 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 GSK3b phosphorylation at Ser9,

indicating that the inhibition of Akt is partial. High concen-

trations of C4 were toxic and did not allow us to test this

further. The observation that C4 prevents PP splitting, a pheno-

typic 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.

Notes

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 Paıtre 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 Concertees

02/07-276, and by the Fondation Medicale Reine Elisabeth, all from

Belgium. Conflict of Interest: None declared.

Address correspondence to email: Andre.Goffinet@dene.ucl.ac.be.

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