Neuronal migration in the murine rostral migratory stream requires serum response factor

Abstract

The central nervous system is fundamentally dependent on guided cell migration, both during development and in adulthood. We report an absolute requirement of the transcription factor serum response factor (SRF) for neuronal migration in the mouse forebrain. Conditional, late-prenatal deletion of Srf causes neurons to accumulate ectopically at the subventricular zone (SVZ), a prime neurogenic region in the brain. SRF-deficient cells of the SVZ exhibit impaired tangential chain migration along the rostral migratory stream into the olfactory bulb. SVZ explants display retarded chain migration in vitro. Regarding target genes, SRF deficiency impairs expression of the beta-actin and gelsolin genes, accompanied by reduced cytoskeletal actin fiber density. At the posttranslational level, cofilin, a key regulator of actin dynamics, displays dramatically elevated inhibitory phosphorylation at Ser-3. Our studies indicate that SRF-controlled gene expression directs both the structure and dynamics of the actin microfilament, thereby determining cell-autonomous neuronal migration.

Fig. 1.

Ablation of SRF in forebrains of Srf^{CamKIIα-iCre} mice causes neuroanatomical defects. Coronal forebrain sections of P17 control (A, C, E, G, and I) and Srf^{CamKIIα-iCre} (B, D, F, H, and K) mice stained with an anti-SRF antibody (A-F) or with Nissl stain (G-K). Only control animals display strong nuclear staining of SRF protein in the CA1, CA3, and dentate gyrus (DG) regions of the hippocampus (C versus D) or in the striatum (E versus F). Nissl staining reveals a missing corpus callosum (indicated by arrows) (G versus H) and a deformed hippocampus in Srf^{CamKIIα-iCre} mice (I versus K). (Scale bars, 500 μm.) Cx, cortex; Hc, hippocampus; St, striatum.

Fig. 2.

SRF-deficient forebrains display cellular accumulation at the SVZ, paralleled by impaired tangential chain migration along the RMS. (A and B) Nissl-stained coronal sections at the striatal plane show abnormal morphology of striatum (St), lateral septum (Se), anterior commissures (Ac), and SVZ in mutant mice. (C and D) Close-up view of the severely broadened SVZ (bSVZ) in brains of Srf^{CamKIIα-iCre} mice compared with the SVZ of control mice. (E and F) Nissl-stained sagittal sections showing the SVZ of control and the bSVZ of mutant mice. The RMS is clearly visible in control mice. (G and H) Staining with an anti-doublecortin antiserum demonstrates a striking retention of neuroblasts in the bSVZ of Srf^{CamKIIα-iCre} mutants. (I and K) BrdUrd labeling reveals an apparent impairment of the ability of neuroblasts born in the SVZ to migrate into the olfactory bulb. (Scale bars, 500 μm.) Ac, anterior commissure; Se, lateral septum; St, striatum.

Fig. 3.

BrdUrd labeling and Matrigel assays demonstrate a defect in neuronal cell migration. (A-F) Three posterior-to-anterior serial coronal sections each of control (A, C, and E) and mutant (B, D, and F) brains were stained with anti-BrdUrd antibody. Labeled cells inside the dashed rectangles were counted (sums of A, C, and E and B, D, and F were each taken as 100%, respectively) and cell numbers in each individual frame are expressed as percentages of the total number of labeled cells per corresponding genotype. Shown are brain sections from the region of the SVZ/bSVZ (12% and 65.8%) (A and B), the region of the RMS (9.2% and 4.2%) (C and D), and the olfactory bulb (78.8% and 30%) (E and F). Note that in brains of mutant mice the majority of labeled cells are still retained in the bSVZ. (G and H) Matrigel assays using cell aggregates dissected from the lateral wall of the lateral ventricles from P6 control (G) and mutant (H) mice, demonstrating impairment in neuronal chain migration in mutant tissue. (Scale bars, 500 μmin A-F and 200 μmin G and H.)

Fig. 4.

Detection of actin and gelsolin proteins by immunohistochemistry and Western blotting. (A-D) Actin stainings (red) were performed on ultrathin Lowicryl sections (100 nm) of dentate gyrus regions (A and B) or the striatum (C and D). Because of the thinness of sections, only F-actin is detected. Nuclei are stained blue. The sections used in C and D are derived from striatal regions also investigated in Fig. 2 (C and D). (E and F) Staining of dentate gyrus regions with an anti-gelsolin antibody. (G) Western blot with 2 μg of forebrain protein extracts of control (c) and mutant (mt) mice probed with an anti-actin antibody. (H) Western blot with 5 μg of protein extracts from microdissected dentate gyrus of three control (c) and three mutant (mt) mice. Comparable loading of proteins in G and H is confirmed by GAPDH or α-tubulin staining. (Scale bars, 20 μmin A-D and 50 μmin E and F.)

Fig. 5.

SRF deficiency causes reduced expression of gelsolin mRNA and elevated inhibitory phosphorylation of cofilin at Ser-3. (A) Pairwise littermate comparison of relative gelsolin mRNA expression as determined by real-time PCR in the hippocampus of five control and five mutant mice. Each column represents the mean of three different cDNA syntheses, followed by PCR. Error bars correspond to standard deviation. (B and C) Real-time PCR analysis of gelsolin (B) and cofilin (C) mRNA expression in 100 Srf(-/-) ES cells, transiently transfected with SRF-VP16 or SRFΔM-VP16 expression plasmids. Primer sequences are given in Supporting Text. RNA was prepared and analyzed 72 h after transfection (^*, P < 0.05, Student's t test). (D) Western blot with 50 μg of protein extracts from control (c) and mutant (mt) animals, derived from cerebral cortex (n = 4), hippocampus (n = 6), and liver (n = 2), probed with antibodies for cofilin and phosphocofilin. Comparable loading of protein is confirmed by GAPDH staining.