Galpha12/Galpha13 deficiency causes localized overmigration of neurons in the developing cerebral and cerebellar cortices

Abstract

The heterotrimeric G proteins G(12) and G(13) link G-protein-coupled receptors to the regulation of the actin cytoskeleton and the induction of actomyosin-based cellular contractility. Here we show that conditional ablation of the genes encoding the alpha-subunits of G(12) and G(13) in the nervous system results in neuronal ectopia of the cerebral and cerebellar cortices due to overmigration of cortical plate neurons and cerebellar Purkinje cells, respectively. The organization of the radial glia and the basal lamina was not disturbed, and the Cajal-Retzius cell layer had formed normally in mutant mice. Embryonic cortical neurons lacking G(12)/G(13) were unable to retract their neurites in response to lysophosphatidic acid and sphingosine-1-phosphate, indicating that they had lost the ability to respond to repulsive mediators acting via G-protein-coupled receptors. Our data indicate that G(12)/G(13)-coupled receptors mediate stop signals and are required for the proper positioning of migrating cortical plate neurons and Purkinje cells during development.

Figures

FIG. 1.

Postnatal lethality and cortical dysplasia in nestin-Cre; Gna12−/−; Gna13flox/flox mice. (A) Western blot analysis of Gα12, Gα13, and Gαq/Gα11 expression in brain extracts prepared from wild-type (WT) and nestin-Cre; Gna12−/−; Gna13flox/flox (KO) mice. (B) Postnatal survival of nestin-Cre; Gna12−/−; Gna13flox/flox, nestin-Cre; Gna12−/+; Gna13flox/flox, nestin-Cre; Gna12−/−; Gna13+/flox, and Gna12−/−; Gna13flox/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−/−; Gna13flox/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−/−; Gna13flox/flox (KO) P21 brains stained with an anti-NeuN antibody. Scale bars are 250 μm (panel C) and 125 μm (panel D).

FIG. 2.

Development of malformations of the cerebellar cortex of nestin-Cre; Gna12−/−; Gna13flox/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−/−; Gna13flox/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−/−; Gna13flox/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−/−; Gna13flox/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).

FIG. 3.

Development of cortical ectopia in nestin-Cre; Gna12−/−; Gna13flox/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).

FIG. 4.

Immunohistochemical analysis of the cerebral ectopia in nestin-Cre; Gna12−/−; Gna13flox/flox embryos. (A and B) Cerebral cortices of E15.5 mice. nestin-Cre; Gna12−/−; Gna13flox/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−/−; Gna13flox/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).

FIG. 5.

Neuronal migration in the cerebral cortex of nestin-Cre; Gna12−/−; Gna13flox/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−/−; Gna13flox/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).

FIG. 6.

Cortical ectopia in NEX-Cre; Gna12−/−; Gna13flox/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).

FIG. 7.

Effect of LPA and S1P on neurite morphology of wild-type and nestin-Cre; Gna12−/−; Gna13flox/flox neurons. (A and B) Wild-type (WT) and nestin-Cre; Gna12−/−; Gna13flox/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−/−; Gna13flox/flox (KO; C and D), or NEX-Cre; Gna12−/−; Gna13flox/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 + Gα13) indicates LPA-induced neurite retraction in cortical neurons transfected with a plasmid encoding Gα13. 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−/−; Gna13flox/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.