Doublecortin is necessary for the migration of adult subventricular zone cells from neurospheres

Abstract

Mutations in human doublecortin (DCX) and knockdown of Dcx in rodents cause radial migration defects in the embryonic cerebral cortex. However, the brain phenotype of Dcx knockout mice is largely normal suggesting that Dcx is not necessary for most migration events. Adult subventricular zone (SVZ) cells migrate tangentially in the rostral migratory stream to the olfactory bulbs. Dcx is expressed in the SVZ but it is unknown if it is necessary for migration. We show that Dcx RNAi reduced SVZ cell migration in vitro, both cell autonomously and non-cell autonomously. Overexpression of Dcx rescued migration after knockdown, but did not increase migration by itself. Thus, Dcx is necessary not only for embryonic radial migration but also migration of adult SVZ cells.

Introduction

Doublecortin is a 40-kDa protein specifically expressed in the leading processes of migrating neurons (Friocourt et al., 2003). Although DCX functions as a microtubule-associated protein (MAP) (Francis et al., 1999, Gleeson et al., 1999), its specific mechanism of action in neuronal migration is still poorly understood. Mutations in the human DCX gene (which is X-linked) cause lissencephaly in males and a less severe phenotype, subcortical laminar heterotopia (SCLH), in heterozygous females (des Portes et al., 1998b, Gleeson et al., 1998). Because of random X-inactivation in females, migrating neurons are separated according to which copy of DCX is active. Therefore, in SCLH, the cerebral cortex appears normal, but a second layer of cortical neurons remain in the subcortical white matter. The hippocampus of both heterozygous females and hemizygous male mice with a targeted mutation in the Dcx gene shows mildly disrupted lamination in the CA3 region (Corbo et al., 2002). Nevertheless, the cerebral cortex and other brain areas, including the olfactory bulb, appear to be normal, suggesting that Dcx is not necessary for all types of neuronal migration and that similar genes have redundant functions (Corbo et al., 2002, Deuel et al., 2006, Koizumi et al., 2006).

Interestingly, when Dcx levels are knocked down at E14 with in utero electroporation of short hairpin RNAs (shRNAs) targeted to Dcx mRNA, radial migration of cortical neurons decreases (Bai et al., 2003). Four days after Dcx RNAi, the majority of electroporated cortical neurons have prematurely terminated migration within the intermediate zone. They remain there, and postnatally produce the characteristic band of ectopic neurons seen in SCLH. Dcx also seems to modulate cell-to-cell communication; neurons that did not take up the shRNA plasmids were unable to migrate through the field of cells affected by RNAi, but were able to migrate past regions with normal cells (Bai et al., 2003). This suggests that Dcx works both cell autonomously and in a non-cell autonomous fashion.

Dcx is expressed in the adult SVZ by neuroblasts that migrate to the olfactory bulbs (Gleeson et al., 1999, Nacher et al., 2001, Yang et al., 2004). The olfactory bulb is normal in the Dcx knockout mouse, suggesting that Dcx is not necessary for SVZ motility. Dcx is also expressed in the neurogenic dentate gyrus and in the piriform cortex (Nacher et al., 2001), regions in which there is little to no migration, suggesting that it may have functions not related to motility. Adult SVZ cell migration is different from embryonic radial migration in the cerebral cortex in a number of ways. (1) SVZ cells migrate much longer distances on average (Lois and Alvarez-Buylla, 1994). (2) SVZ neuroblasts migrate as longitudinal arrays of cells (chains), whereas cortical neurons migrate individually (Lois et al., 1996). (3) SVZ cells migrate through a meshwork of specialized astrocyte processes and extracellular matrix (tangential migration); cortical migration occurs along radial glia (radial migration) (Thomas et al., 1996). (4) SVZ neuroblasts are capable of proliferation, but migrating cortical neurons are postmitotic (Thomas et al., 1996). SVZ cells can be cultured as floating neurospheres which, when plated migrate away from the neurospheres. Therefore, we decided to use this system to ask if Dcx knockdown also diminishes SVZ cell migration. We cultured SVZ cells as neurospheres from adult mice and showed that knockdown of Dcx by RNAi significantly decreases migration of cells, in both a cell autonomous and non cell-autonomous fashion.