Rac1 controls the formation of midline commissures and the competency of tangential migration in ventral telencephalic neurons - PubMed (original) (raw)

Comparative Study

Rac1 controls the formation of midline commissures and the competency of tangential migration in ventral telencephalic neurons

Lei Chen et al. J Neurosci. 2007.

Abstract

Previous studies using dominant-mutant constructs have implicated Rac1 GTPase in neuritogenesis and neuronal migration. However, overexpression of dominant mutants generally blocks Rho-GTPase activity; thus, it may not reveal the specific or physiological functions of Rac1. To address this issue, we have applied a conditional gene-targeting strategy, using Foxg1-Cre mice to delete Rac1 in the ventricular zone (VZ) of telencephalon and Dlx5/6-Cre-IRES (internal ribosomal entry site)-EGFP (enhanced green fluorescent protein) (Dlx5/6-CIE) in the subventricular zone (SVZ) of ventral telencephalon, respectively. Surprisingly, the deletion of Rac1 in VZ progenitors did not prevent axonal outgrowth of telencephalic neurons. However, the anterior commissure was absent, and the corpus callosal as well as hippocampal commissural axons failed to cross the midline in Rac1/Foxg1-Cre knock-out embryos. The thalamocortical and corticothalamic axons also showed defasciculation or projection defects. These results suggest that Rac1 controls axon guidance rather than neuritogenesis. In addition, although Rac1/Foxg1-Cre knock-out embryos showed delayed radial migration of cortical projection neurons and severe impairment of tangential migration by the ventral telencephalon-derived interneurons, deletion of Rac1 in the SVZ by Dlx5/6-CIE mice produced no discernible defects in tangential migration. These contrasting effects of Rac1 deletion on tangential migration suggest that Rac1 is dispensable for cellular motility per se during neuronal migration. Together, these results underscore the challenge of deciphering the biological functions of Rac1, and Rho-GTPases in general, during mammalian brain development. Moreover, they indicate that Rac1 has a critical role in axon guidance and in acquisition of migratory competency during differentiation of the progenitors for the ventral telencephalon-derived interneurons.

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Figures

Figure 1.

Generation of Foxg1–Cre-driven, forebrain-specific Rac1 conditional knock-out mice. A, Schematic representations of the floxed Rac1 locus before and after the Cre-mediated recombination. Also indicated are the PCR products of wild-type (Rac1WT), floxed (Rac1flox), and recombined–floxed (Rac1Δflox) alleles of Rac1. B, Comparison of PCR genotyping analysis using genomic DNA collected from the tail or the forebrain of embryos of different genotypes. Note that the presence of Foxg1Cre/+ transgene converts Rac1flox into the Rac1Δflox allele in genomic DNA in the forebrain but not the tail. C, Immunoblot analysis of Rac1, Rac3, and Cdc42 in the E18.5 forebrain lysates of wild-type (WT), Rac1+/flox; Foxg1Cre/+ (Rac1+/flox), and Rac1flox/flox; Foxg1Cre/+ (Rac1flox/flox) embryos. Actin was used as a loading control. This assay shows that Rac1 is specifically deleted in the Rac1flox/flox; Foxg1Cre/+ embryo without changing the protein levels of Rac3 and Cdc42.

Figure 2.

Commissural tract defects in the Rac1-mutant (Rac1flox/flox; Foxg1Cre/+) embryos. A, B, Nissl stain shows the abnormalities of E18.5 Rac1-mutant embryo including enlarged ventricles, the absence of AC, irregular cortical lamination, and the disappearance of a distinct SP layer, as well as a thicker fornix (F) bundle. C, D, Immunostaining for the L1 cell-adhesion molecule confirms the absence of AC and reduction of the IC fibers in the Rac1-mutant embryos. E–H, The L1 immunostain also confirms midline-crossing defects in the CC and HC fibers in the forebrain. Ctx, Cortex. Scale bar (in H): A–D, 200 μm; E–H, 20 μm.

Figure 3.

Three-dimensional analysis of major axon tract defects in the telencephalic Rac1-deleted embryos. A–J, E18.5 control (Rac1+/flox) and Rac1-mutant (Rac1flox/flox; Foxg1Cre/+) embryos were analyzed by T2-weighted MRI and 3D reconstruction for visualization in the transverse (A–D), coronal (E–H), and sagittal (I, J) axes. Note the absence of AC and uncrossed CC as well as HC axons in the Rac1-mutant embryos. There was also an increase in thickness of the fornix (F) bundle in the Rac1-mutant embryos. H, Rac1-deficient embryos.

Figure 4.

Rac1 deletion causes defects of thalamocortical and corticothalamic connections. A, B, L1 immunostaining shows the thalamocortical axons have ascended into the developing cortical plate (CP) in control (Rac1+/flox) embryos at E18.5, whereas their counterparts in the Rac1-mutant (Rac1flox/flox; Foxg1Cre/+) embryos are halted in the intermediate zone (IZ). C, D, TGA1 immunostaining shows the formation of corticothalamic axons (arrows) in the base of developing cerebrum of both control and Rac1-mutant embryos at E16.5. St, Striatum. E–H, Crystals of DiI placed in the lateral cerebral cortex (Ctx) label the efferent fibers into the thalamus (Th) in E18.5 control embryos (E) but not in Rac1-mutant embryos (F). Scale bar (in F): A, B, 40 μm; C, D, 200 μm; E, F, 400 μm.

Figure 5.

Neurite formation in the absence of Rac1 GTPase. A, PCR analysis shows the floxed Rac1 allele is near-completely replaced by the recombined–floxed allele of Rac1 (Rac1Δflox) in the telencephalic neurons cultured from E14.5 Rac1flox/flox; Foxg1Cre/+) embryos. WT, Wild type. B, Immunoblots show great reduction of the Rac1 protein level, but not those of phospho-PAK, phospho-LIMK, and phospho-MLCK in the neuronal cell lysates. Shown are representatives of three sets of experiments. C, Histogram of the average numbers of primary and secondary neurite in Rac1+/fox and Rac1-mutant (Rac1flox/flox; Foxg1Cre/+) neurons. Rac1-mutant neurons have a significantly increased complexity of the neurite formation (p < 0.01 by t test). D, E, Immunostaining of β-tubulin III of Rac1+/fox and Rac1-mutant neurons at 2 DIV. F, G, Double labeling of anti-β-tubulin III (green) and phalloidin (red) shows that F-actin is also concentrated in the growth cones of control and Rac1-mutant neurons. H, I, Immunostaining of β-tubulin III of control and Rac1-mutant neurons at 4 DIV. Note the extension of axons (arrow) in neurons of both genotypes. Scale bar (in I): D, E, 80 μm; F, G, 20 μm; H, I, 100 μm.

Figure 6.

Preserved radial migration of cortical neurons in the absence of Rac1 GTPase. A, B, Birth-dating analysis with BrdU injected at E10.5 and E11.5 and the embryos examined at E17.5. Similar to control embryos, the majority of BrdU-positive, VI/SP neurons (highlighted by white lines) are located in the intermediate zone (IZ) of both control (Rac1+/flox) and Rac1-mutant (Rac1flox/flox; Foxg1Cre/+) embryos. MZ, Marginal zone. C–E, Birth-dating analysis with BrdU injected at E15.5 and the embryos examination at E18.5. The number of BrdU-positive cells in four equal sectors (I–IV) of the cortex were quantified for comparison (3 embryos for each genotype and a total of 5640 cells were counted to derive the histogram). The percentage of BrdU-positive cells is significantly increased in the bottom quarter (I) and decreased in the top quarter (IV) of developing cerebral cortex in Rac1-mutatnt embryos (*p < 0.01 by t test). F, G, Immunostaining of Brn1, a marker of neurons destined for the upper cortical layer, shows more positively labeled cells in the IZ in E18.5 Rac1-mutant embryos compared with age-matched control embryos. This pattern is consistent with delayed migration of cortical neurons. Scale bar (in G): A–D, 200 μm; F, G, 40 μm.

Figure 7.

Abnormal distribution of ventral telencephalon-derived interneurons in Rac1flox/flox; Foxg1Cre/+ embryos. A, B, The olfactory bulbs (arrow) were disproportionately small in E16.5 to E18.5 Rac1flox/flox; Foxg1Cre/+ embryos compared with littermates. C, D, Immunohistochemistry for GAD67 shows the LGE-derived GABAergic interneurons are markedly decreased in the olfactory bulb of Rac1-mutant embryos. GL, Glomerular layer; GCL, granule cell layer. E, F, In contrast, GAD67-positive cells were detected in the basal forebrain of both control and Rac1-mutant embryos. G–J, In situ hybridization of Lhx6.1 shows equal formation of GABAergic interneurons in the MGE (small arrow) of control and Rac1-mutant embryos at E14.5. However, Lhx6.1-expressing cells are halted in the mantle region of MGE (large arrow) in the Rac1-mutant embryos at E16.5 (J), when compared with control littermate embryos (I). K, L, Immunostaining shows many GAD67-positive cells, a marker for cortical interneurons, in the cerebral cortex of E18.5 control embryos (K), but not in Rac1-mutant embryos (L). Scale bar (in L): C, D, 100 μm; E–J, 400 μm; K, L, 40 μm.

Figure 8.

Normal distribution of ventral telencephalon-derived interneurons in Rac1flox/flox; Dlx5/6–CIE embryos. A, Immunostaining showed the expression of EGFP (used here as a surrogate marker of Cre recombinase) in the SVZ, but not the VZ, of presumptive LGE and MGE in E10.5 Dlx5/6–CIE embryos. B, Comparison of EGFP expression, propidium iodide-stained, and merged images indicates the expression of Cre throughout the entire ventral telencephalon, except the VZ, in E14.5 Dlx5/6–CIE embryos. C, PCR analysis shows the conversion of a floxed Rac1 (Rac1flox) allele into recombined–floxed allele (Rac1Δflox) in the striatum of E18.5 Rac1flox/flox; Dlx5/6-CIE embryos. D, E, Nissl stain shows the apparently normal morphology of E18.5 Rac1/Dlx5/6–CIE embryo, when compared with control heterozygous littermate. F–M, Comparison of ventral telencephalon-derived neurons between control and Rac1/Dlx5/6–CIE embryos or 12-d-old (P12) mice. There was no obvious difference in the number and distribution of GAD67-positive or EGFP-positive cells in the E18.5 olfactory bulbs (F, G) and cerebral cortex (H, I), respectively. Similarly, there is no obvious difference of parvalbumin-positive (J, K), or calbindin-positive (L, M) cells in the P12 cerebral cortex (Ctx) between control and Rac1/Dlx5/6–CIE animals. Scale bar (in M): A, 50 μm; B, 400 μm; D, E, 200 μm; F, G, 100 μm; H, I, 50 μm; J–M, 400 μm.

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Rac1 controls the formation of midline commissures and the competency of tangential migration in ventral telencephalic neurons - PubMed (original) (raw)