G{alpha}12/G{alpha}13 Deficiency Causes Localized Overmigration of Neurons in the Developing Cerebral and Cerebellar Cortices

The heterotrimeric G proteins G₁₂ and G₁₃ link G-protein-coupledreceptors to the regulation of the actin cytoskeleton and theinduction of actomyosin-based cellular contractility. Here weshow that conditional ablation of the genes encoding the ${alpha}$ -subunitsof G₁₂ and G₁₃ in the nervous system results in neuronal ectopiaof the cerebral and cerebellar cortices due to overmigrationof cortical plate neurons and cerebellar Purkinje cells, respectively.The organization of the radial glia and the basal lamina wasnot disturbed, and the Cajal-Retzius cell layer had formed normallyin mutant mice. Embryonic cortical neurons lacking G₁₂/G₁₃ wereunable to retract their neurites in response to lysophosphatidicacid and sphingosine-1-phosphate, indicating that they had lostthe ability to respond to repulsive mediators acting via G-protein-coupledreceptors. Our data indicate that G₁₂/G₁₃-coupled receptorsmediate stop signals and are required for the proper positioningof migrating cortical plate neurons and Purkinje cells duringdevelopment.

INTRODUCTION

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INTRODUCTION

The cortical structures of the adult mammalian central nervoussystem are organized into cell layers. This laminar organizationis the result of the precisely controlled migration of cellsfrom distinct germinal layers to their final destination inthe developing cortex (18, 20, 33, 48, 52). The mechanisms underlyingthe well orchestrated formation of cortical structures are onlypartially understood. Some of the molecules regulating the correctmigration of neurons in the developing cortex have been discoveredby analyzing mutations that disrupt neuronal migration and formationof neuronal layers (3, 9, 19). One of the best studied signalingsystems involved in the proper positioning of neurons in thedeveloping cerebral and cerebellar cortices consists of theextracellular protein reelin and its receptors VLDLR and ApoER2(22, 42, 50). However, a variety of other proteins like integrinsor β-amyloid precursor proteins have been shown to be requiredfor the correct layering of cortical neurons (2, 12, 21), indicatingthat multiple mechanisms are involved in the migration and layeringof neurons during cortical development (3, 19).

The intracellular signaling cascades involved in cell migrationhave been studied in various cells including neurons and fibroblasts(11, 19, 39, 41, 43). Monomeric GTPases of the Rho family havebeen shown to play a central role in the regulation of cytoskeletalrearrangements and regulation of adhesive functions underlyingcell migration (43). While Rac and Cdc42 are involved in theformation of cell protrusions and the formation of adhesions,RhoA plays an important role in the retraction of cell protrusions.Multiple G-protein-coupled receptors have been shown to regulatethe migratory activity of cells by modulating the activity ofRac/Cdc42 and/or Rho (44, 54). Activation of RhoA via G-protein-coupledreceptors is primarily mediated by the ubiquitously expressedheterotrimeric G proteins G₁₂ and G₁₃ (5, 44). Mice lackingthe ${alpha}$ -subunit of G₁₃ die in utero (38), whereas G ${alpha}$ ₁₂-deficientmice are viable (13). In the present study we generated conditionalmouse mutants carrying inactivating mutations in the genes codingfor G ${alpha}$ ₁₂ (Gna12) and G ${alpha}$ ₁₃ (Gna13) in the nervous system in orderto study their role in neural development. Our study revealedan unexpected role of the G₁₂/G₁₃-mediated signaling pathwayin the development of the cerebral and cerebellar cortices.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Mice. Mice in which the gene coding for G ${alpha}$ ₁₃, Gna13, is flanked byloxP sites (Gna13^flox) (35) were crossed to the constitutivelyG ${alpha}$ ₁₂-deficient mouse line (13) and to mice which express therecombinase Cre under the control of the nestin promoter (51,55) or the NEX promoter (10). Genotyping for the Gna13^flox andGna12^– alleles was described previously (35). The primersused for detecting the nestin-Cre transgene were 5'-AGTGCTGACTCTCCTCGGCTT-3'and 5'-CCAGACCTGTTCCACCTCTG-3'. Mice were housed under specific-pathogen-freeconditions, and all animal experiments were performed in accordancewith institutional animal care and use committee regulations.The genetic background of the mice was predominantly C57BL6/N(at least a sixth-generation backcross), and littermates withthe nestin-Cre; Gna13^fl/fl; Gna12^+/+, Gna13^fl/fl; Gna12^–^/^–,or Gna13^fl/fl; Gna12^–^/+ genotype were used as controls.

Western blot analysis. Brain lysates were subjected to sodium dodecyl sulfate-polyacrylamidegel electrophoresis. After blotting, nitrocellulose membraneswere probed with antibodies against G ${alpha}$ ₁₂ (30), G ${alpha}$ ₁₃, or G ${alpha}$ _q/11(Santa Cruz Biotechnology).

Histology. Mice were deeply anesthetized with pentobarbital (100 mg/kgof body weight, intraperitoneally) and perfused with 4% paraformaldehydevia the left cardiac ventricle. For histology, whole heads ordissected brains were postfixed overnight and then stored in0.5% paraformaldehyde at 4°C for Vibratome sectioning orembedding in paraffin. Alternatively, fixed tissues were incubatedfor 24 h in 30% sucrose-phosphate-buffered saline (PBS) at 4°Cbefore being frozen on dry ice. Vibratome sections (50 µm)were cut, paraffin-embedded material was sectioned at 6 µm,and frozen brains were sectioned at 20 µm. Sections werestained with cresyl violet following a standard protocol.

Immunohistochemistry. Immunohistochemistry on Vibratome sections and paraffin-embeddedmaterial was performed with the following antibodies: anti-NeuN(Chemicon; 1:2,000) and anticalbindin (Chemicon; 1:2,000). Immunohistochemistryon paraffin-embedded material was performed with the followingantibodies: anticalretinin (Chemicon; 1:250), antireelin (Chemicon;clone G10 at 1:50), and antibromodeoxyuridine (anti-BrdU) (Sigma;1:100) diluted in PBS. Incubation with first antibodies wasperformed for 24 h at 4°C. For secondary antibodies, weused rabbit anti-mouse immunoglobulin G (Dako; diluted 1:50in PBS) for 1 h at room temperature. Bound secondary antibodieswere detected by the avidin-biotin peroxidase complex (VectorLaboratories, Burlingame, CA) and visualized with diaminobenzidine(Vector Laboratories). For anti-BrdU stainings, fluorescentlylabeled secondary antibodies were used (Dianova).

Immunohistochemistry on frozen sections was performed afterfixation in 4% (wt/vol) paraformaldehyde in PBS (pH 7.4) overnight,followed by 30 min of incubation in blocking buffer (10% normalgoat serum and 0.5% Triton X-100 in PBS). The first antibodyincubation with anticalretinin (Chemicon; 1:500), anti-reelin(Chemicon; clone G10 at 1:500), and anti-RC2 antisera (1:100)was carried out overnight at 4°C at the indicated dilutionsfollowed by incubation (1 h) with the secondary antibodies,which were conjugated either to Alexa TM 594 (red fluorescence)or Alexa TM 488 (green fluorescence) (Molecular Probes, Eugene,OR). Nuclei were counterstained with DAPI (4',6'-diamidino-2-phenylindole;Sigma). Fluorescent specimens were viewed with a Nikon C1siconfocal laser-scanning microscope.

BrdU birthdating and morphometric analysis. Pregnant females were injected at 11:00 a.m. either on embryonicday 12.5 (E12.5) or on E15.5, with a single dose of BrdU (Sigma)at a concentration of 100 µg/g of body weight. Femaleswere killed 24 h later on E18.5, and pups were genotyped byPCR as described above. Brains from pups were processed forparaffin embedding, and incorporated BrdU was detected by immunohistochemistry(see above). Cells with dense staining of more than half ofthe nucleus were considered BrdU positive.

Primary cultures from mouse cortex. Cultures of cortical and cerebellar neurons were prepared withsome minor modifications according to the procedure describedby Banker and Goslin (1) for hippocampal neurons. Cerebral andcerebellar cortices from E16.5 or E17.5 mouse embryos were preparedin ice-cold dissection solution (PBS with 30 mM HEPES and 33mM glucose, pH 7.38) and washed once in PBS. After incubationwith 0.05% trypsin (Invitrogen) for 15 min at 37°C, thetissues were washed with PBS and triturated with fire-polishedPasteur pipettes in plating medium (minimal essential mediumsupplemented with 10% fetal bovine serum, 1 mM sodium pyruvate[Invitrogen], 25 mM glucose, and 25 µM glutamic acid [Sigma]).Cells were seeded in plating medium on poly-L-lysine (Sigma)-coatedglass slides (Nunc) or plastic dishes (Falcon) at a densityof 1.3 x 10⁵ cells/well ( $~$ 7.3 x 10⁴ cells/cm², for live-cellimaging) or 1 x 10⁶ cells/well ( $~$ 1 x 10⁵ cells/cm², for RhoAactivation assay), respectively, and maintained at 37°Cin 5% CO₂. After 4 h, plating medium was replaced by growthmedium (neurobasal medium with B-27 supplement and 0.5 mM L-glutamine[Invitrogen]), and the cells were cultured for 20 h withoutchanging the medium. Transfection of primary neurons with aeukaryotic expression plasmid encoding G ${alpha}$ ₁₃ was performed withan Amaxa (Cologne, Germany) mouse neuron transfection kit accordingto the manufacturer's instructions.

Live-cell imaging. Live-cell images were recorded on a Leica DM IRE2 microscopeequipped with a 37°C/5% CO₂ environmental control chamberusing a Leica DC 350 FX camera and Leica FW4000 software. Randomlyselected cerebral and cerebellar neurons were imaged beforeand 5, 15, and 20 min after addition of 10 µM lysophosphatidicacid (LPA) and 1 µM sphingosine-1-phosphate (S1P) (Biomol)or 20 min after addition of 2 µg/ml ephrin-A5-Fc (R&DSystems) preclustered with 20 µg/ml anti-human immunoglobulinG-Fc (Sigma) as described previously (28). Cerebellar neuronalcells were acetone-fixed and stained with an anticalbindin antibody(Chemicon; 1:2,000) to identify Purkinje cells. Neurite retraction(percent of retracted neurites) was calculated from 15 to 45cells per embryo, and the number of embryos analyzed was 3 to6.

Determination of RhoA activity. RhoA activation assay was performed using a G-Lisa RhoA activationassay kit (Cytoskeleton) according to the manufacturer's instructions.Briefly, cells were incubated for 30 s with vehicle alone (PBS)or 10 µM LPA, immediately washed with ice-cold PBS, andprepared in ice-cold lysis buffer. Lysates were incubated inmicroplate wells coated with a RhoA-GTP-binding protein andthe bound active RhoA was detected using a RhoA-specific antibodyand chemoluminescence.

Statistical analysis. The statistical analyses were performed using the Mann-WhitneyU test.

RESULTS

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RESULTS

To analyze the role of the G₁₂/G₁₃-mediated signaling pathwayin the development of the nervous system, we crossed G ${alpha}$ ₁₂-deficientmice which were homozygous for a floxed G ${alpha}$ ₁₃ allele (35) witha transgenic mouse line expressing Cre under the control ofthe neuron-specific enhancer of the nestin promoter (51). Thenestin-Cre allele has been shown to result in a very efficientrecombination in neuronal and glial precursor cells startingat E10.5. Nonneuronal/nonglial cells of the nervous system,like those of blood vessels and meninges, are not recombined(12, 55). When nestin-Cre; Gna12^–^/+; Gna13^flox/flox micewere crossed with Gna12^–^/+; Gna13^flox/flox animals, allgenotypical combinations were obtained in the living offspringwith the expected frequencies. Western blot analyses of whole-brainlysates of nestin-Cre; Gna12^–^/^–; Gna13^flox/floxmice showed the absence of both G ${alpha}$ ₁₂ and G ${alpha}$ ₁₃ proteins (Fig. 1A).The postnatal growth of nestin-Cre; Gna12^–^/^–; Gna13^flox/floxmice was retarded and resulted in premature death between postnatalday 10 (P10) and P40 (Fig. 1B). At about 2 weeks of age, survivingdouble mutants exhibited obvious abnormalities like a reducedbody size and ataxia. In contrast, mice with one intact Gna12or Gna13 allele (nestin-Cre; Gna12^–^/+; Gna13^flox/floxor nestin-Cre; Gna12^–^/^–; Gna13^+/flox) showed normalpostnatal development and had a normal life expectancy. Whenbrains of P21 nestin-Cre; Gna12^–^/^–; Gna13^flox/floxmice were analyzed histologically, severe malformations of theircerebellar and cerebral cortices were observed in all animalsanalyzed (23) (Fig. 1C and D). The cerebellar cortex of therostral part of the vermis was highly disorganized, while thecaudal part and the hemispheres appeared to be grossly normal(Fig. 1C). The cerebral cortices of double knockouts were convolutedand marked by invasions of neurons into layer I as well as byectopia (Fig. 1D). The cortical structures of mice carryingone intact Gna12 allele (nestin-Cre; Gna12^–^/+; Gna13^flox/flox)or one intact Gna13 allele (nestin-Cre; Gna12^–^/^–;Gna13^+/flox) were morphologically normal (data not shown). Thisindicates that the loss of all four alleles of the Gna12 andGna13 genes is required for the observed phenotypical abnormalities.

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FIG. 1. Postnatal lethality and cortical dysplasia in nestin-Cre; Gna12^–^/^–; Gna13^flox/flox mice. (A) Western blot analysis of G ${alpha}$ ₁₂, G ${alpha}$ ₁₃, and G ${alpha}$ _q/G ${alpha}$ ₁₁ expression in brain extracts prepared from wild-type (WT) and nestin-Cre; Gna12^–^/^–; Gna13^flox/flox (KO) mice. (B) Postnatal survival of nestin-Cre; Gna12^–^/^–; Gna13^flox/flox, nestin-Cre; Gna12^–^/+; Gna13^flox/flox, nestin-Cre; Gna12^–^/^–; Gna13^+/flox, and Gna12^–^/^–; Gna13^flox/flox mice; the total number of analyzed animals was 54, 51, 38, and 29, respectively. (C) Sagittal section through the cerebellum of wild type (WT) and nestin-Cre; Gna12^–^/^–; Gna13^flox/flox (KO) animals at P21 stained with an anti-NeuN antibody. Cortical malformations in the knockout cerebella were restricted to the rostral part of the vermis. (D) Frontal sections of the cerebral cortex from wild type (WT) and nestin-Cre; Gna12^–^/^–; Gna13^flox/flox (KO) P21 brains stained with an anti-NeuN antibody. Scale bars are 250 µm (panel C) and 125 µm (panel D).

In the postnatal rostral cerebellum, the normal lobulation wasabsent. Instead, the cortex appeared to be thinner and convolutedwith heterotopia and ectopia. Despite these severe irregularities,Purkinje cells had basically developed their dendritic tree,which filled the molecular layer, and granule cells had migratedinward past the Purkinje cell layer and had formed an innergranule cell layer (Fig. 2A to C). To determine the developmentalstage during which the severe malformation of the rostral cerebellarcortex started, we analyzed serial sagittal sections at P7,P4, and P0 as well as at E18.5, E17.5, E15.5, and E13.5. Visualizationof Purkinje cells by staining of sections for calbindin showeda severe mislocalization of Purkinje cells already at P4 andP0, indicating that Purkinje cells of the rostral cerebellarcortex had migrated abnormally (Fig. 2D to G). The first signsof abnormal Purkinje cell migration could be observed at E17.5.Purkinje cells of the developing anterior vermis migrated intothe external granular layer (EGL) and formed nests of cellsbelow the pia mater. A transmigration of Purkinje cells intothe tectum was not observed (Fig. 2H to L). The overmigrationof Purkinje cells into the EGL of the anterior vermis of thecerebellum was not accompanied by any obvious change in theEGL structure or by alterations in the architecture of the radialglia fibers (Fig. 2M to O).

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FIG. 2. Development of malformations of the cerebellar cortex of nestin-Cre; Gna12^–^/^–; Gna13^flox/flox mice. (A to C) Sagittal section through the rostral part of a mutant cerebellum at P21 stained with cresyl violet and an anticalbindin antibody. The high-power images (B and C; boxes in panel A) show that despite the severe disorganization of the rostral cerebellar cortex, the basal lamination into an internal granule cell layer, Purkinje cell layer, and molecular layer was largely intact. (D to G) Sagittal sections of wild-type (D and F) and nestin-Cre; Gna12^–^/^–; Gna13^flox/flox (E and G) mice at P4 (D and E) and at P0 (F and G) stained with anticalbindin antibody. Sections are shown with the rostral part of the cerebellum facing the left side. (H to L) Sagittal sections of a wild-type (WT) (H and I) and a nestin-Cre; Gna12^–^/^–; Gna13^flox/flox (KO) cerebellum (J to L) at E17.5 stained with cresyl violet and an anticalbindin antibody. While the granule cell layer and the Purkinje cell layer are clearly separated in the wild type, clusters of Purkinje cells invade the external granule cell layer in mutant cerebella (arrow) and nests of Purkinje cells can be seen in the EGL (arrowhead). The EGL is marked by dotted red lines. (M to O) Sagittal section of a nestin-Cre; Gna12^–^/^–; Gna13^flox/flox cerebellum at E18.5 stained with anti-calbindin (red) and anti-RC2 antibodies (radial glia; green). Arrows in panel M indicate overmigrating Purkinje cells entering the EGL. r, rostral; c, caudal. Scale bars are 500 µm (A), 31.25 µm (B and C), 250 µm (D, E, H, J, and M to O), 125 µm (F and G), and 25 µm (I, K, and L).

In order to determine the time point at which the first obviousdefects in the development of the cerebral cortex occurred,nestin-Cre; Gna12^–^/^–; Gna13^flox/flox animals wereanalyzed at different stages before and after birth. The earliesttime point at which careful analysis of the cerebral morphologyof mutant animals consistently revealed abnormalities was E15.5.At this stage, cells migrated at some places into the marginalzone (Fig. 3A to D). Two days later, ectopic clusters of cellswhich had migrated through the marginal zone could be seen inthe subarachnoidal space (Fig. 3E to H). Ectopic clusters werepreferentially found in the frontal areas of the cortex. Neuronswere the major constituent of the ectopia, as demonstrated bystaining with anti-NeuN antibodies (data not shown).

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FIG. 3. Development of cortical ectopia in nestin-Cre; Gna12^–^/^–; Gna13^flox/flox embryos. Shown are coronal sections of wild-type (WT) (A, B, E, and F) and mutant (KO) (C, D, G, and H) cortices at E15.5 (A to D) and E17.5 (E to H). Overmigration of cortical plate neurons in mutant cortices was first seen at E15.5 (arrow in C and D). At E17.5, huge areas of ectopic neurons had formed which filled the whole molecular layer and reached into the subarachnoidal space (indicated by stars in G and H). MZ, marginal zone; CP, cortical plate. Scale bars are 125 µm (A, C, F, and H), 62.5 µm (B and D), and 250 µm (E and G).

To further characterize the cortical defects in nestin-Cre;Gna12^–^/^–; Gna13^flox/flox animals, we analyzed variouscomponents of the cortex at different stages during development.To test whether defects in the basal lamina preceded the neuronalovermigration, we stained the developing cortex with anti-lamininantibodies at E14.5, a time point at which no ectopia couldbe observed in nestin-Cre; Gna12^–^/^–; Gna13^flox/floxanimals, as well as at E15.5, the earliest time point when overmigrationof cortical plate neurons could be seen. Despite an extensiveanalysis of the basal lamina morphology at E14.5, no defectswere observed at this stage (data not shown). At E15.5, thebasal lamina in regions of overmigrating neurons appeared alsoto be normal (Fig. 4A). At E16.5, when neuronal ectopia wasmore prominent, some fragmentation of the basal lamina couldbe observed (Fig. 4C). Scattered fragments of laminin couldalso be seen on the surface of the ectopia (Fig. 4C).

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FIG. 4. Immunohistochemical analysis of the cerebral ectopia in nestin-Cre; Gna12^–^/^–; Gna13^flox/flox embryos. (A and B) Cerebral cortices of E15.5 mice. nestin-Cre; Gna12^–^/^–; Gna13^flox/flox embryos were sectioned coronally and stained with antilaminin (A) or anticalretinin (B) antibodies. Sections were counterstained with DAPI. Shown are areas similar to those shown in Fig. 3C and D in which cortical plate neurons have invaded the molecular layer (arrows). Boxes indicate magnified areas, broken lines mark the outer border of the cortical plate. (C to E) Cerebral cortices of E16.5 wild-type (WT) and nestin-Cre; Gna12^–^/^–; Gna13^flox/flox (KO) embryos were sectioned coronally and stained with antilaminin (C, red), anti-RC2 (C and D, green), or anticalretinin (D and E, red) antibodies. Sections were counterstained with DAPI. Shown are representative areas of mutant cortices with ectopic neurons (marked by stars) and corresponding regions of cortices from wild-type embryos. Boxes indicate magnified areas. Bar lengths are 125 µm (upper panels in A to E), 41.5 µm (lower panels in A and B), and 62.5 µm (lower panels in C to E).

Cajal-Retzius cells which secrete reelin have been shown toplay an important role in the regulation of neuronal migrationin the developing cortex (34, 42, 47). Staining of Cajal-Retziuscells with an antibody directed against calretinin in areasin which cortical plate neurons started to invade the molecularlayer at E15.5 showed that the number and localization of Cajal-Retziuscells were not altered (Fig. 4B). By using anti-reelin and anti-calbindinantibodies, we observed that Cajal-Retzius cells and reelinwere still present on the surface of neuronal ectopia (Fig.4D and E). These findings indicate that overmigrating neuronsdo not move through the Cajal-Retzius cell layer and suggestthat a defect in the proper localization and function of Cajal-Retziuscells is unlikely to cause the overmigration of cortical neuronsin nestin-Cre; Gna12^–^/^–; Gna13^flox/flox mice.

To analyze the morphology of radial glia, we immunostained embryoniccortical sections with an antibody directed against the radialglia marker RC2. Radial glia fibers showed a parallel organizationboth in wild-type as well as in mutant cortices (Fig. 4C and D).In regions with ectopia, radial glia fibers often reached intothe ectopic clusters of neurons, suggesting that a detachmentof radial glia fibers from the basal membrane is unlikely tohave caused the formation of ectopia.

In order to determine the time point when ectopic neurons wereborn, we labeled proliferating cells with BrdU at E12.5 andE15.5 (Fig. 5). Areas of the cortex which did not show any obviousmalformations were not significantly different from wild-typecortices with regard to the number or distribution of BrdU-positivecells at E18.5 (data not shown). This indicates that there wereno general defects in neuronal proliferation or survival innestin-Cre; Gna12^–^/^–; Gna13^flox/flox mice. Whenectopic neurons were studied at E18.5, cells which were labeledat E12.5 as well as cells labeled at E15.5 were found in theectopia (Fig. 5). Thus, in regions developing neuronal ectopia,neurons born at different stages of cortical development showalterations in migratory behavior, whereas no major layeringdefects could be observed in normal areas of the cortex.

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FIG. 5. Neuronal migration in the cerebral cortex of nestin-Cre; Gna12^–^/^–; Gna13^flox/flox embryos. Pregnant mice were injected at E12.5 (A) or E15.5 (B) with BrdU, and the distribution of BrdU-labeled neurons was determined in wild-type (WT) and nestin-Cre; Gna12^–^/^–; Gna13^flox/flox (KO) embryos at E18.5. Neurons born at both E12.5 and E15.5 took part in the formation of neuronal ectopia. The broken lines mark the surface of the developing brain. Areas containing ectopic neurons are marked by stars. Bar lengths are 125 µm (upper panels in A and B) and 62.5 µm (lower panels in A and B).

Since G ${alpha}$ ₁₂/G ${alpha}$ ₁₃ are ubiquitously expressed G protein ${alpha}$ -subunitsand since in nestin-Cre; Gna12^–^/^–; Gna13^flox/floxmice G ${alpha}$ ₁₂/G ${alpha}$ ₁₃ deficiency is induced both in neural and glialprecursor cells, we cannot exclude that the lack of G₁₂/G₁₃-mediatedsignaling in radial glia cells rather than in migrating postmitoticneurons is responsible for the observed defects. To clarifythis uncertainty, we used mice in which Cre expression is drivenby the NEX promoter, which restricts recombination to principalneurons of the forebrain excluding glial cells and interneurons(10). Similar to nestin- Cre; Gna12^–^/^–; Gna13^flox/floxmice, NEX-Cre; Gna12^–^/^–; Gna13^flox/flox mice displayedmultiple ectopia of the cerebral cortices (Fig. 6), thus stronglysuggesting that lack of G ${alpha}$ ₁₂/G ${alpha}$ ₁₃ in cortical neurons is responsiblefor the observed neuronal overmigration.

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FIG. 6. Cortical ectopia in NEX-Cre; Gna12^–^/^–; Gna13^flox/flox animals. Shown are coronal sections of wild-type (A and B) and mutant (KO) (C and D) cortices at P21. Areas containing ectopic neurons are marked by stars. Scale bars are 250 µm (A and C) and 125 µm (B and D).

Based on the morphological analysis of the cerebral and cerebellarcortices, the overmigration of radially migrating neurons appearsto be the earliest defect during the development of corticalstructures in nestin-Cre; Gna12^–^/^–; Gna13^flox/floxembryos. We therefore hypothesize that Purkinje cells as wellas neurons of the cerebral cortex which lack G ${alpha}$ ₁₂/G ${alpha}$ ₁₃ are unableto receive stop signals which under wild-type conditions ensurethat neurons do not migrate into the EGL of the cerebellar cortexor into the marginal zone of the cerebral cortex. This hypothesiswould be consistent with the cellular role of G₁₂/G₁₃, whichhave been shown to mediate receptor-dependent retraction ofneurites and neurite-like structures (26, 29, 46), a processwhich would be expected to inhibit migratory activity. Lysophospholipidslike LPA and S1P have been shown to induce neurite retractionby activation of G-protein-coupled receptors and RhoA (36) andhave been demonstrated to regulate cortical development (23).To test whether cerebellar Purkinje cells and cortical neuronsfrom nestin-Cre; Gna12^–^/^–; Gna13^flox/flox embryoswere still able to respond with morphological changes to LPAand S1P, we isolated Purkinje cells and cortical neurons atE17.5. While both LPA and S1P induced neurite retraction inwild-type neurons (24, 40, 45, 49) (Fig. 7A to D), Purkinjecells and cortical neurons prepared from E17.5 nestin-Cre; Gna12^–^/^–;Gna13^flox/flox mice were completely unresponsive (Fig. 7A to D).The inability of G ${alpha}$ ₁₂/G ${alpha}$ ₁₃-deficient neurons to retract theirneurites in response to LPA was accompanied by a lack of LPA-inducedRhoA activation, while wild-type neurons showed robust activationof RhoA in response to LPA (Fig. 7E). The lack of LPA effectson cortical neurons was not due to a general defect in G ${alpha}$ ₁₂/G ${alpha}$ ₁₃-deficientneurons, since neurite retraction in response to ephrin-A5 wasnot affected by the lack of G₁₂/G₁₃-mediated signaling (Fig.7F and G). The defect in LPA-induced neurite retraction in corticalneurons from NEX-Cre; Gna12^–^/^–; Gna13^flox/flox micecould be rescued by the transfection of cells with a eukaryoticexpression plasmid encoding G ${alpha}$ ₁₃ (Fig. 7F and G). These dataindicate that the retraction of neuronal processes induced bymediators acting through G-protein-coupled receptors is mediatedby the G₁₂/G₁₃-Rho pathway and that defects in G-protein-coupledreceptor-dependent retraction of cellular processes may underliethe observed phenotype of mice lacking G ${alpha}$ ₁₂/G ${alpha}$ ₁₃ in the nervoussystem.

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FIG. 7. Effect of LPA and S1P on neurite morphology of wild-type and nestin-Cre; Gna12^–^/^–; Gna13^flox/flox neurons. (A and B) Wild-type (WT) and nestin-Cre; Gna12^–^/^–; Gna13^flox/flox (KO) neurons from E17.5 cerebellar cortices were studied by live-cell imaging before (–LPA) and after (+LPA) addition of LPA. Thereafter, cells were stained for calbindin (anti-Calb.) to identify embryonic cerebellar Purkinje cells. Shown are representative images of embryonic Purkinje cells (A) as well as a statistical evaluation of neurite retraction in response to LPA (B). (C to G) Cortical plate neurons were isolated from wild-type (WT), nestin-Cre; Gna12^–^/^–; Gna13^flox/flox (KO; C and D), or NEX-Cre; Gna12^–^/^–; Gna13^flox/flox (KO; E to G) E16.5 embryonic brains. Isolated cells were then monitored by live-cell imaging before (–LPA/–S1P) or after addition of LPA (+LPA), S1P (+S1P), or ephrin-A5 (EphA5) as indicated. The gray bar in panel G (KO + G ${alpha}$ ₁₃) indicates LPA-induced neurite retraction in cortical neurons transfected with a plasmid encoding G ${alpha}$ ₁₃. Shown are representative images (C and F) as well as a statistical evaluation of the effects of 10 µM LPA, 1 µM S1P, and 2 µg/ml ephrin-A5 on neurite retraction in wild-type and mutant cortical plate neurons (D and G). Fifteen to forty-five cells per embryo were analyzed, and the total number of embryos per experiment was three to six. (E) Effect of 10 µM LPA on RhoA activity in cortical neurons from wild-type (WT) or NEX-Cre; Gna12^–^/^–; Gna13^flox/flox (KO) E16.5 embryonic brains. Shown is the RhoA activation relative to the basal activity (=1). All values are means ± standard deviations; *, P < 0.05; **, P < 0.01; n.s., not significant.

DISCUSSION

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DISCUSSION

In this study we analyzed the consequences of G ${alpha}$ ₁₂/G ${alpha}$ ₁₃ doubledeficiency for the development of the mammalian brain. We reportthat lack of G ${alpha}$ ₁₂/G ${alpha}$ ₁₃ in cortical neurons results in the overmigrationof cerebellar Purkinje cells and of postmitotic neurons of thecerebrum, resulting in the formation of ectopia and severe malformationsof cortical structures. This indicates that G-protein-coupledreceptors acting via G₁₂/G₁₃ are involved in the proper positioningof radially migrating cortical neurons.

Under normal conditions, radially migrating cortical neuronsstop at defined sites. The signals which induce a stop of migrationare not well defined. One of the best characterized factorsregulating neuronal migration in the cortex is the extracellularprotein reelin, which is produced by Cajal-Retzius cells (42,50). Mislocalization of Cajal-Retzius cells and reelin expressionhave been described in several mutants with cortical ectopia(12, 16, 37). In contrast to these mutants, we found that bothCajal-Retzius cells and reelin showed normal localization atearly stages of neuronal overmigration and were still coveringthe surface of ectopic clusters of neurons, suggesting thata defect in reelin function is unlikely to have caused the corticaldefects in nestin-Cre; Gna12^–^/^–; Gna13^flox/floxembryos.

The radial glia fibers along which cortical neurons migrateoutward are attached at the basal lamina, which is producedby meningeal fibroblasts. Defects in several proteins whichare required for the proper assembly of the basal lamina orfor the anchoring of the radial glia end feet have been shownto lead to defects in neuronal migration, resulting in ectopia(2, 8, 12, 15, 16, 21, 37). These migratory effects have beensuggested to represent secondary effects due to the misplacementof Cajal-Retzius cells (4, 16, 17, 37, 53). In the nestin-Cre;Gna12^–^/^–; Gna13^flox/flox embryos, the basic architectureof radial glia fibers appeared to be undisturbed. In addition,the basal lamina appeared to be intact in areas where overmigrationof neurons occurred at later stages of development. Once cellshad overmigrated and had formed ectopic cell clusters, the basallamina was rarefied or fragmented, which most likely resultedfrom the expansion of the cortical surface in ectopic areas.This strongly indicates that defects in the structure of thebasal lamina or the radial glia are not primary causes of thedefects in cortical development of nestin-Cre; Gna12^–^/^–;Gna13^flox/flox mice.

Based on the fact that G₁₂/G₁₃ couple receptors to the activationof RhoA and the stimulation of actomyosin-based contractility(5, 44), it is likely that the loss of this regulatory pathwayinterferes with the normal regulation of cell migration andmay cause the observed overmigration phenotype. Studies of migratoryprocesses in neurons and other cells have demonstrated thatmovement occurs by extension of a leading process, formationof new adhesion junctions, cell body contraction, and detachmentof adhesions at the cell rear (7, 20, 31). These processes areregulated by various signaling pathways which involve Rho familyGTPases (41, 43). While Rac and Cdc42 are responsible for theformation of the leading process, RhoA is required for the rearend contraction. In neurons, neurite-like protrusions whichresemble the leading process of migrating neurons are retractedwhen RhoA is activated (6, 11, 14, 25, 32), and RhoA activationin leading processes of migrating neurons would be expectedto be incompatible with proper migration and to act as a stopsignal.

Several mediators such as the lysophospholipids S1P and LPAhave been shown to induce neurite retraction via the activationof G-protein-coupled receptors and the activation of RhoA (23,24, 29, 40). Consistent with this, we found that both LPA andS1P induced neurite retraction in embryonic cortical neuronsand cerebellar Purkinje cells, whereas these effects were abrogatedin neurons lacking G ${alpha}$ ₁₂/G ${alpha}$ ₁₃. The fact that mice lacking one ortwo of the various LPA and S1P receptors do not show overmigrationof cortical neurons (23, 27) indicates that the lysophospholipidsLPA or S1P alone are obviously not critically involved in theregulation of neuronal migration in the cortex. Nevertheless,our data suggest that mediators acting through G₁₂/G₁₃-coupledreceptors act as stop signals for migrating postmitotic corticalneurons and thereby contribute to the establishment of properlylaminated cerebellar and cerebral cortices.

ACKNOWLEDGMENTS

We thank Melanie Bernhard for technical assistance, AndreasFaissner and Alexander von Holst for kindly providing the anti-RC2antiserum, Elisabeth Pollerberg for help with the transfectionof neurons, and Ulrike Engel and Christian Ackermann from theNikon Imaging Center at the University of Heidelberg for experttechnical support.

This study was supported by the Collaborative Research Center488 (SFB 488) of the German Research Foundation.

FOOTNOTES

* Corresponding author. Mailing address: Institute of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany. Phone: 49-6221-54-8246. Fax: 49-6221-54-8549. E-mail: Stefan.Offermanns{at}pharma.uni-heidelberg.de

Published ahead of print on 17 December 2007.

These authors contributed equally.

REFERENCES

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